Branched polyalkylene glycols

ABSTRACT

The present invention provides a branched polyalkylene glycol wherein three or more single-chain polyalkylene glycols and a group having reactivity with an amino acid side chain, the N-terminal amino group or the C-terminal carboxyl group in a polypeptide or a group convertible into the group having reactivity are bound; and a physiologically active polypeptide modified with the branched polyalkylene glycol.

TECHNICAL FIELD

The present invention relates to polyalkylene glycols having a branchedstructure which are useful as modifiers for polypeptides having aphysiological activity (physiologically active polypeptides) and tophysiologically active polypeptides modified with the polyalkyleneglycols. The present invention also relates to pharmaceuticalcompositions comprising the physiologically active polypeptides modifiedwith the polyalkylene glycols.

BACKGROUND ART

Physiologically active polypeptides are useful as therapeutic agents forspecific diseases. However, they are unstable when administered intoblood, and a sufficient pharmacological effect can rarely be expected.For instance, physiologically active polypeptides having a molecularweight of less than 60,000 administered into blood are mostly excretedinto urine by renal glomerular filtration, and their use as therapeuticagents is not expected to give a significant therapeutic effect andoften requires repeated administration. Some other physiologicallyactive polypeptides are degraded by hydrolases and the like existing inblood, thereby losing their physiological activities. Further, someexogenous physiologically active polypeptides have physiologicalactivities effective for the treatment of diseases, but it is known thatsuch exogenous physiologically active polypeptides and physiologicallyactive polypeptides produced by recombinant DNA techniques sometimesinduce immunoreaction when administered into blood to cause seriousside-effects such as anaphylactic shock owing to the difference instructure between them and endogenous physiologically activepolypeptides. In addition, some physiologically active polypeptides havephysical properties unsuitable for use as therapeutic agents, e.g. poorsolubility.

One of the known attempts to solve these problems in usingphysiologically active polypeptides as therapeutic agents is tochemically bind at least one molecule of an inactive polymer chain tophysiologically active polypeptides. In many cases, desirable propertiesare conferred on the polypeptides or proteins by chemically bindingpolyalkylene glycols such as polyethylene glycol to them.

For example, superoxide dismutase (SOD) modified with polyethyleneglycol has a remarkably prolonged half-life in blood and shows a durableaction [Pharm. Res. Commun., Vol. 19, p. 287 (1987)]. There is also areport of modification of granulocyte colony-stimulating factor (G-CSF)with polyethylene glycol [J. Biochem., Vol. 115, p. 814 (1994)]. GillianE. Francis, et al. summarized examples of polyethylene glycol-modifiedpolypeptides such as asparaginase, glutaminase, adenosine deaminase anduricase [Pharm. Biotechnol., Vol. 3, Stability of ProteinPharmaceuticals, Part B, p. 235 (1992), Plenum Press, New York].Further, it is known that modification of physiologically activepolypeptides with polyalkylene glycols give effects such as enhancementof thermal stability [Seibutsubutsuri (Biophysics), Vol. 38, p. 208(1998)] and solubilization in organic solvents [Biochem. Biophys. Res.Commun.: BBRC, Vol. 122, p. 845 (1984)].

With regard to the methods for binding polyalkylene glycols to peptidesor proteins, it is known to introduce an active ester of carboxylicacid, a maleimido group, a carbonate, cyanuric chloride, a formyl group,an oxiranyl group or the like to an end of a polyalkylene glycol andbind it to an amino group or a thiol group in a polypeptide[Bioconjugate Chem., Vol. 6, p. 150 (1995)]. These techniques includethe binding of a polyethylene glycol to a specific amino acid residue ina physiologically active polypeptide, which causes enhancement ofstability in blood without impairing the biological activities of thepeptide or protein. Examples of the polyethylene glycol modificationspecific to amino acid residues in physiologically active polypeptidesinclude the binding of a polyethylene glycol to the carboxyl terminus ofa growth hormone-releasing factor through norleucine as a spacer [J.Peptide Res., Vol. 49, p. 527 (1997)] and the specific binding of apolyethylene glycol to cysteine introduced to the 3-position ofinterleukin-2 by recombinant DNA techniques [BIO/TECHNOLOGY, Vol. 8, p.343 (1990)].

Many of the above polyalkylene glycol-modified polypeptides are obtainedby binding of linear polyalkylene glycols. However, it has been foundthat binding of branched polyalkylene glycols is preferable forobtaining chemically modified polypeptides having a high activity. It isgenerally known that the durability of a chemically modified polypeptidein blood is increased as the molecular weight of a polyalkylene glycolis higher or the modification ratio higher [J. Biol. Chem., Vol. 263, p.15064 (1988)], but in some cases, the physiological activity of aphysiologically active polypeptide is impaired by raising themodification ratio. This is partly because a specific amino group orthiol group in the physiologically active polypeptide which is necessaryfor its physiological activity is modified with a chemical modifier. Forexample, it is known that the physiological activity of interleukin-15lowers according to the modification ratio [J. Biol. Chem., Vol. 272, p.2312 (1997)].

On the other hand, it is difficult to synthesize high molecular weightpolyalkylene glycols having a uniform molecular weight distribution anda high purity. In the case of monomethoxypolyethylene glycols, forexample, contamination with diol components as impurities is known.Accordingly, attempts have been made to prepare high molecular weightmodifiers by branching currently available polyalkylene glycols having anarrow molecular weight distribution and a high purity. Such attemptsprovide chemically modified polypeptides having a high physiologicalactivity with a high durability retained even with a decreasedmodification ratio. Further, it is considered that a larger part of thesurface of molecules of physiologically active polypeptides can becovered with polyalkylene glycols by branching of the polyalkyleneglycols. For example, double-chain polyethylene glycol derivativesprepared by using cyanuric chloride as the group having a branchedstructure are known (Japanese Published Unexamined Patent ApplicationsNos. 72469/91 and 95200/91). In this case, a methoxypolyethylene glycolhaving an average molecular weight of 5,000 is utilized, but thiscompound has the problem of toxicity due to the triazine ring. JapanesePublished Unexamined Patent Application No. 153088/89 discloses that achemically modified polypeptide having a high activity can be obtainedfrom a comb-shaped polyethylene glycol which is a copolymer ofpolyethylene glycol and maleic anhydride at a lower modification ratiocompared with a linear polyethylene glycol. However, this compound hasmany reactive sites for a polypeptide, which causes impairment of thephysiological activity of a physiologically active polypeptide, and hasan ununiform molecular weight distribution. Also known are a compoundhaving two polyethylene glycol chains through a benzene ring prepared byusing cinnamic acid as a material (Japanese Published Unexamined PatentApplication No. 88822/91) and compounds having two polyethylene glycolchains prepared by using lysine as a material (WO96/21469, U.S. Pat. No.5,643,575).

As illustrated by the above examples, compounds having two polyalkyleneglycol chains are known, but those having three or more polyalkyleneglycol chains have not been produced. Although U.S. Pat. No. 5,643,575suggests a three-branched, water-soluble, non-antigenic polymer, itcontains no disclosure of the method for producing the three-branchedcompound or of specific examples and provides no information about theexcellent effect of the three-branched compound.

There exists a need for a chemically modified polypeptide with improveddurability which retains the activity of the physiologically activepolypeptide and whose renal glomerular filtration is suppressed. Inorder to produce the chemically modified polypeptide exhibiting suchproperties, there is also a need for a modifier with a low toxicity andan improved stability which has an excellent molecular size-increasingeffect and a narrow and uniform molecular weight distribution.

DISCLOSURE OF THE INVENTION

An object of the present invention is to provide, as a chemical modifierfor a physiologically active polypeptide, a branched chemical modifierhaving polyalkylene glycol chains which has an excellent molecularsize-increasing effect. Another object of the present invention is toprovide a physiologically active polypeptide modified with the branchedpolyalkylene glycol.

The present inventors made intensive studies on branched polyalkyleneglycol modifying reagents having a novel structure for modification ofphysiologically active polypeptides. As a result, the inventors havefound that modifying reagents having a molecular size-increasing effectsuperior to that of known linear or double-chain polyalkylene glycolscan be obtained by preparing modifiers having three or more polyalkyleneglycol chains. They have further found that modification ofphysiologically active polypeptides with the above branched polyalkyleneglycols gives physiologically active polypeptides modified with branchedpolyalkylene glycols having three or more chains whose renal glomerularfiltration is suppressed to a degree beyond expectation and whosedurability in blood is remarkably improved compared with those modifiedwith known linear or double-chain polyalkylene glycols, while retainingtheir physiological activities.

It has thus been found that the above branched polyalkylene glycols areexcellent chemical modifiers and the present invention has beencompleted.

That is, the present invention provides a branched polyalkylene glycolwherein three or more single-chain polyalkylene glycols and a grouphaving reactivity with an amino acid side chain, the N-terminal aminogroup or the C-terminal carboxyl group in a polypeptide or a groupconvertible into the group having reactivity are bound sumultaneously; aphysiologically active polypeptide or its derivative modified with thepolyalkylene glycol; and a pharmaceutical composition or a therapeuticagent comprising the physiologically active polypeptide or itsderivative modified with the polyalkylene glycol. From anotherviewpoint, the present invention relates to a chemically modifiedpolypeptide wherein a physiologically active polypeptide or itsderivative is modified with at least one polyalkylene glycol mentionedabove directly or through a spacer; and a pharmaceutical composition ora therapeutic agent comprising the chemically modified polypeptide.

The present invention is described in detail below.

The branched polyalkylene glycols of the present invention include anybranched polyalkylene glycols wherein three or more single-chainpolyalkylene glycols and a group having reactivity with an amino acidside chain, the N-terminal amino group or the C-terminal carboxyl groupin a polypeptide or a group convertible into the group having reactivityare bound. Preferred branched polyalkylene glycols are those whereinthree or more single-chain polyalkylene glycols and one to three groupshaving reactivity with an amino acid side chain, the N-terminal aminogroup or the C-terminal carboxyl group in a polypeptide or one to threegroups convertible into the groups having reactivity are bound. Morepreferred are branched polyalkylene glycols wherein three or foursingle-chain polyalkylene glycols and one group having reactivity withan amino acid side chain, the N-terminal amino group or the C-terminalcarboxyl group in a polypeptide or one group convertible into the grouphaving reactivity are bound.

The single-chain polyalkylene glycol may be any single-chainpolyalkylene glycol but is preferably R¹—M_(n)—X¹ (in which M, n, R¹ andX¹ have the same meanings as defined below).

The group having reactivity with an amino acid side chain, theN-terminal amino group or the C-terminal carboxyl group in a polypeptideor a group convertible into the group having reactivity may be any grouphaving reactivity with an amino acid side chain, the N-terminal aminogroup or the C-terminal carboxyl group in a polypeptide or any groupconvertible into the group having reactivity.

Preferred branched polyalkylene glycols of the present invention includecompounds represented by formula (I):(R¹—M_(n)—X¹)_(m)L(X²—X³—R²)_(q)  (I){wherein L represents a group capable of having four or more branches;

-   M represents OCH₂CH₂, OCH₂CH₂CH₂, OCH(CH₃)CH₂,    (OCH₂CH₂)_(r)—(OCH₂CH₂CH₂)_(s) (in which r and s, which may be the    same or different, each represent an arbitrary positive integer) or    (OCH₂CH₂)_(ra)—[OCH(CH₃) CH₂]_(sa) (in which ra and sa have the same    meanings as the above r and s, respectively);-   n represents an arbitrary positive integer;-   m represents an integer of 3 or more;-   q represents an integer of 1 to 3;-   R¹ represents a hydrogen atom, lower alkyl or lower alkanoyl;-   R² represents a group having reactivity with an amino acid side    chain, the N-terminal amino group or the C-terminal carboxyl group    in a polypeptide or a group convertible into the group having    reactivity;-   X¹ represents a bond, O, S, alkylene, (OCH₂)_(ta) (in which ta    represents an integer of 1 to 8), (CH₂)_(tb)O (in which tb has the    same meaning as the above ta), NR³ (in which R³ represents a    hydrogen atom or lower alkyl), R⁴—NH—C(═O)—R⁵ [in which R⁴    represents a bond, alkylene or O(CH₂)_(tc) (in which tc has the same    meaning as the above ta) and R⁵ represents a bond, alkylene or    OR^(5a) (in which R^(5a) represents a bond or alkylene)],    R⁶—C(═O)—NH—R⁷ [in which R⁶ represents a bond, alkylene or R^(6a)O    (in which R^(6a) has the same meaning as the above R^(5a)) and R⁷    represents a bond, alkylene or (CH₂)_(td)O (in which td has the same    meaning as the above ta)], R⁸—C(═O)—O (in which R⁸ has the same    meaning as the above R^(5a)) or O—C(═O)—R⁹ (in which R⁹ has the same    meaning as the above R^(5a))-   X² represents a bond, 0 or (CH₂)_(te)O (in which te has the same    meaning as the above ta);-   X³ represents a bond or alkylene; and three or more R¹—M_(n)—X¹'s    may be the same or different, and when two or three X²—X³—R²'s are    present (when q is 2 or 3), they may be the same or different}-   [hereinafter the compounds represented by formula (I) are referred    to as Compounds (I), and the same shall apply to the compounds of    other formula numbers].

In the definitions of the groups in formula (I), the lower alkyl and thelower alkyl moiety of the lower alkanoyl include linear or branchedalkyl groups having 1 to 8 carbon atoms such as methyl, ethyl, n-propyl,isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, neopentyl,hexyl, heptyl and octyl. The alkylene includes alkylene groups having 1to 8 carbon atoms such as methylene, ethylene, n-propylene,isopropylene, n-butylene, isobutylene, sec-butylene, tert-butylene,pentylene, neopentylene, hexylene, heptylene and octylene.

In formula (I), M represents OCH₂CH₂, OCH₂CH₂CH₂, OCH(CH₃) CH₂,(OCH₂CH₂)_(r)—(OCH₂CH₂CH₂)_(s) (in which r and s, which may be the sameor different, each represent an arbitrary positive integer) or(OCH₂CH₂)_(ra)—[OCH(CH₃) CH₂] _(sa) (in which ra and sa have the samemeanings as the above r and s, respectively), and when M is(OCH₂CH₂)_(ra)—(OCH₂CH₂CH₂)_(s) (in which r and s have the same meaningsas defined above) or (OCH₂CH₂)_(ra)—[OCH(CH₃)CH₂]_(sa) (in which ra andsa have the same meanings as defined above), r and s, and ra and sa arepreferably 1 to 100,000, more preferably 1 to 1,000.

In formula (I), n represents an arbitrary positive integer and ispreferably 10 to 100,000, more preferably 100 to 1,000.

The average molecular weight of the polyalkylene glycol moietyrepresented by M_(n) is preferably ca. 1,000 to 1,000,000, morepreferably 5,000 to 100,000. When Mn is —(OCH₂CH₂)_(n)—, it is preferredthat polyethylene glycols used as starting materials are monodisperseand their molecular weight distribution represented by Mw(weight-average molecular weight)/Mn (number-average molecular weight)is 1.1 or less, and commercially available ones can be utilized whenthose having an average molecular weight of 30,000 or less are required.For example, monomethoxypolyethylene glycols having an average molecularweight of 2,000, 5,000, 10,000, 12,000, 20,000 or the like can be used.

In formula (I), q represents an integer of 1 to 3 and is preferably 1.

In formula (I), m represents an integer of 3 or more and is preferably 3to 4.

The molecular weight of the branched polyalkylene glycols represented byformula (I) is preferably in the range of 500 to 1,000,000.

In formula (I), L represents a group capable of having four or morebranches and may have a hydroxyl group, substituted or unsubstitutedlower alkyl, lower alkoxy, amino, carboxy, cyano, formyl or the like asa substituent thereon. The lower alkyl and the lower alkyl moiety of thelower alkoxy have the same meaning as the above lower alkyl, and thesubstituent in the substituted lower alkyl includes a hydroxyl group,amino, lower alkanoyloxy, lower alkanoylamino, lower alkoxy, loweralkoxyalkoxy, lower alkanoyl, lower alkoxycarbonyl, loweralkylcarbamoyl, lower alkylcarbamoyloxy and the like. The lower alkylmoiety of the lower alkanoyloxy, the lower alkanoylamino, the loweralkoxy, the lower alkoxyalkoxy, the lower alkanoyl, the loweralkoxycarbonyl, the lower alkylcarbamoyl and the lower alkylcarbamoyloxyhas the same meaning as the above lower alkyl.

As the group capable of having four or more branches represented by L,any group can be used so far as it is capable of binding to a groupconvertible into a group having reactivity with an amino acid sidechain, the N-terminal amino group or the C-terminal carboxyl group in apolypeptide or the group having reactivity through X²—X³, and is capableof having as branches three or more molecules of single-chainpolyalkylene glycols through X¹. Examples of L include groups formed byremoving four or more hydrogen atoms from a polyol or a polycarboxylicacid having a molecular weight of 1,000 or less. Examples of the polyolinclude low molecular compounds such as glucose, D,L-sorbitol, ribose,erythritol, pentaerythritol, tricine(N-[tris(hydroxymethyl)methyl]glycine), inositol, cholic acid,3,4,5-trihydroxybenzoic acid (gallic acid), 2,4,6-trihydroxybenzoicacid, 3,4,5-trihydroxybenzaldehyde, quinic acid, shikimic acid andtris(hydroxymethyl)aminomethane, and stereoisomers thereof. Examples ofthe polycarboxylic acid include low molecular compounds such as1,4,5,8-naphthalenetetracarboxylic acid, pyromellitic acid,diethylenetriaminepentaacetic acid, 1,2,3,4-butanetetracarboxylic acid,citric acid and γ-carboxyglutamic acid, and stereoisomers thereof.

Examples of preferred L include a group formed by removing four or morehydrogen atoms from tricine, a group formed by removing four or morehydrogen atoms from shikimic acid, a group formed by removing four ormore hydrogen atoms from quinic acid, a group formed by removing four ormore hydrogen atoms from erythritol, a group formed by removing four ormore hydrogen atoms from pentaerythritol, and a group formed by removingfour or more hydrogen atoms from glucose.

The structure of the L moiety can be constructed by using a commerciallyavailable compound as it is, using the compound through conversion intoa derivative suitable for the binding of polyalkylene glycols accordingto a general organic synthetic method, or using the compound after theprotection of a functional group [edited by The Chemical Society ofJapan, Jikken Kagaku Koza (Experimental Chemistry Course), fourthedition (1992), Organic Synthesis I-V, Maruzen; PROTECTIVE GROUPS INORGANIC SYNTHESIS, second edition, JOHN WILEY & SONS, INC. (1991); etc.]

Cyclohexanes other than those mentioned above can be synthesizedaccording to the method of Kihi, et al. [Daiyukikagaku (Great OrganicChemistry), Vol. 6, p. 183 (1958), Asakura Shoten], the method of G. E.McCasland and E. Clide Horswill [Journal of American Chemical Society,Vol. 76, p. 2373 (1954)] or the like.

In Compound (I), the binding of polyalkylene glycols to L through X¹ canbe easily effected by combining the reactions known in the generalorganic synthetic methods [edited by The Chemical Society of Japan,Jikken Kagaku Koza (Experimental Chemistry Course), fourth edition, pp.19-23 (1992), Organic Synthesis I-V, Maruzen].

In formula (I), R² represents a group having reactivity with an aminoacid side chain, the N-terminal amino group or the C-terminal carboxylgroup in a polypeptide or a group convertible into the group havingreactivity.

Namely, the above group having reactivity includes groups reactive withany one of the side chains of lysine, cysteine, arginine, histidine,serine, threonine, tryptophan, aspartic acid, glutamic acid, glutamineand the like, the N-terminal amino group and the C-terminal carboxylgroup in a polypeptide. Examples of such groups include a hydroxylgroup, carboxy, formyl, amino, vinylsulfonyl, mercapto, cyano,carbamoyl, halogenated carbonyl, halogenated lower alkyl, isocyanato,isothiocyanato, oxiranyl, lower alkanoyloxy, maleimido,succinimidooxycarbonyl, substituted or unsubstituted aryloxycarbonyl,benzotriazolyloxycarbonyl, phthalimidooxycarbonyl, imidazolylcarbonyl,substituted or unsubstituted lower alkoxycarbonyloxy, substituted orunsubstituted aryloxycarbonyloxy, tresyl, lower alkanoyloxycarbonyl,substituted or unsubstituted aroyloxycarbonyl, substituted orunsubstituted aryldisulfido, and azido.

In the definitions of the above groups, the lower alkyl moiety of thelower alkoxycarbonyloxy, the halogenated lower alkyl, the loweralkanoyloxy and the lower alkanoyloxycarbonyl has the same meaning asthe above lower alkyl. The aryl moiety of the aryloxycarbonyl, thearyloxycarbonyloxy and the aryldisulfido includes aryls having 6 to 14carbon atoms such as phenyl, naphthyl, biphenyl and anthryl. The aroylmoiety of the aroyloxycarbonyl includes aroyls having 7 to 13 carbonatoms such as benzoyl, naphthoyl and phthaloyl. The halogen moiety ofthe halogenated carbonyl and the halogenated lower alkyl includes atomsof fluorine, chlorine, bromine and iodine.

The substituted lower alkoxycarbonyloxy has 1 to 3 substituents whichmay be the same or different. Examples of the substituents are ahydroxyl group, carboxy and halogen. The halogen has the same meaning asdefined above.

The substituted aryloxycarbonyl, the substituted aryloxycarbonyloxy, thesubstituted aryldisulfido and the subsituted aroyloxycarbonyl have 1 to3 substituents which may be the same or different. Examples of thesubstituents are a hydroxyl group, carboxy, halogen, cyano and loweralkyl. The halogen and the lower alkyl have the same meanings as definedabove, respectively.

The group represented by R² may be contained in the starting materialfor constructing the structure of the L moiety, or may be formed byprotecting a necessary functional group in the starting material with anappropriate protective group in advance [PROTECTIVE GROUPS IN ORGANICSYNTHESIS, second edition, JOHN WILEY & SONS, INC. (1991) etc.],removing the protective group after binding polyalkylene glycols to Lthrough X¹'s to make branches, and converting it, if necessary. Further,after polyalkylene glycols are bound to L through X¹'s to make branches,the above R² can also be introduced to L, if necessary through X² or X³,by a usual organic synthetic method.

More specifically, the branched polyalkylene glycols of the presentinvention can be produced, for example, by the following processes. Theprocesses for producing the branched polyalkylene glycols of the presentinvention are not limited thereto.

Process 1: Production of compounds wherein X¹ is a bond, O, alkylene,O(CH₂)_(ta) or (CH₂)_(tb)O

Compound (Ia), i.e. Compound (I) wherein X¹ is a bond, O, alkylene,O(CH₂)_(ta) (in which ta has the same meaning as defined above) or(CH₂)_(tb)O (in which tb has the same meaning as defined above) can beproduced, for example, by the following process.

A polyol having three or more hydroxyl groups is dissolved or suspendedin an appropriate solvent (e.g. N,N-dimethylformamide, dimethylsulfoxide, toluene, tetrahydrofuran, acetonitrile or pyridine) underanhydrous conditions, and 3 mol or more of a halide or tosylate of apolyalkylene glycol or a monoalkyl ether or monocarboxylate esterthereof (hereinafter, they are collectively referred to as polyalkyleneglycol A) is added thereto in the presence of 1 to 30 mol of anappropriate base (e.g. sodium hydride, zinc oxide, sodium hydroxide ortriethylamine), followed by reaction at −20 to 150° C. for 1 hour to 10days to obtain a mixture containing a branched polyalkylene glycolhaving three or more chains.

The polyol is selected from commercially available compounds such asquinic acid, glucose, sorbitol, ribose, erythritol, pentaerythritol,tricine and inositol, and compounds derived from the commerciallyavailable compounds. Examples of the compounds derived from thecommercially available compounds include polyols obtained by reducingpolycarboxylic acid selected from ethylenediaminetetraacetic acid,1,2,4,5-benzenetetracarboxylic acid, γ-carboxyglutamic acid and the likewith an appropriate reducing agent according to a usual organicsynthetic method [edited by The Chemical Society of Japan, Jikken KagakuKoza (Experimental Chemistry Course), fourth edition, Vols. 19-21(1992), Maruzen]. Suitable reducing agents include lithium aluminumhydride, sodium borohydride, sodium cyanoborohydride and hydrogen.

The polyol may have hydroxyl groups at any positions and can be used inthe reaction after appropriate protection of a functional groupunnecessary for the reaction by the method described in PROTECTIVEGROUPS IN ORGANIC SYNTHESIS, second edition, JOHN WILEY & SONS, INC.(1991), etc. or conversion into a derivative.

The halide or tosylate of polyalkylene glycol A can readily be producedby various methods disclosed in a review by Samuel Zalipsky[Bioconjugate Chem., Vol. 6, p. 150 (1995)] and the like. The halide ortosylate of polyalkylene glycol A used for the binding may have anyaverage molecular weight so long as the molecular weight distribution isuniform (preferably Mw/Mn is 1.1 or less).

The obtained mixture containing a branched polyalkylene glycol havingthree or more chains can be used in the next step at the purity as it isor after purifying and isolating the branched polyalkylene glycol havingthree, four, five or more chains to a desired purity according to thenumber of branches by a known method such as ion-exchangechromatography, reversed phase chromatography, hydrophobicchromatography, two-phase partition or recrystallization. By the abovesteps, some of Compounds (Iaj), i.e. Compounds (Ia) wherein R² is ahydroxyl group are obtained.

On the other hand, the desired branched polyalkylene glycol having threeor more chains can also be prepared by using a polyhalide or a polytosyland polyalkylene glycol A. In this case, the desired compound can beobtained by dissolving or suspending 3 molar equivalents or more ofpolyalkylene glycol A in an appropriate solvent (e.g.N,N-dimethylformamide, dimethyl sulfoxide, toluene or tetrahydrofuran),and adding 1 molar equivalent of a polyhalide or polytosyl thereto inthe presence of 1 to 30 mol of an appropriate base (e.g. sodium hydride,zinc oxide, sodium hydroxide or triethylamine) per mol of polyalkyleneglycol A, followed by reaction at −20 to 150° C. for 1 hour to 10 days.

The polyhalide may be a commercially available compound or may beobtained by converting the above polyol into a halide [edited by TheChemical Society of Japan, Jikken Kagaku Koza (Experimental ChemistryCourse), fourth edition, Vol. 19 (1992), Maruzen]. The polytosyl can beobtained by dissolving or suspending the polyol in an appropriatesolvent (e.g. N,N-dimethylformamide, dimethyl sulfoxide, toluene,tetrahydrofuran, acetonitrile or pyridine), and adding thereto 1 to 3molar equivalents (based on the hydroxyl group) of a tosyl halide in thepresence of 1 to 30 mol (based on the hydroxyl group) of an appropriatebase (e.g. sodium hydride, zinc oxide, sodium hydroxide, triethylamineor potassium naphthalene), followed by reaction at −20 to 150° C. for 1hour to several days.

Then, R² is introduced into the obtained mixture containing a branchedpolyalkylene glycol having three or more chains or a compound purifiedtherefrom. As R², a functional group remaining in a polyol, a polyhalideor a polytosyl can be utilized as it is after polyalkylene glycol A or ahalide or tosylate thereof is bound to the polyol, polyhalide orpolytosyl. Alternatively, a functional group bound to a polyol isprotected in advance, and after polyalkylene glycol A or a halide ortosylate thereof is bound, a group obtained by removing the protectinggroup of the functional group may be utilized as R². In this case, afterat least one hydroxyl group or other functional group in the abovepolyol, polyhalide or polytosyl is protected with an appropriateprotective group, polyalkylene glycol A or a halide or tosylate thereofis introduced to the other hydroxyl groups, halogen or tosyl groupmoiety by the same method as above to synthesize a compound with threeor more polyalkylene glycol chains bound, and then the functional groupfrom which the protective group is removed is utilized as such, or atleast one of the functional groups is converted to R² according to themethod described below. The functional groups present in the polyol,polyhalide or polytosyl before or after binding polyalkylene glycol A ora halide or tosylate thereof include carboxy, amino, halogen, cyano,formyl, carbonyl and the like, in addition to a hydroxyl group. As forthe protective groups for functional groups, suitable protective groupsfor a hydroxyl group include benzyl, tert-butyl, acetyl,benzyloxycarbonyl, tert-butyloxycarbonyl, dimethyl-tert-butylsilyl,diphenyl-tert-butylsilyl, trimethylsilyl, triphenylsilyl, tosyl andtetrahydropyranyl; those for amino include methyl, ethyl,9-fluorenylmethyloxycarbonyl, benzyloxycarbonyl, nitrobenzyloxycarbonyl,N-phthalimido, acetyl and tert-butyloxycarbonyl; those for carboxyinclude benzyl, methyl, ethyl, tert-butyl, 9-fluorenylmethyl,methoxyethoxymethyl, 2,2,2-trichloroethyl, 2-(trimethylsilyl)ethyl,cinnamoyl, allyl and nitrophenyl; and those for formyl include dimethylacetal, diethyl acetal, dibenzyl acetal and 1,3-dioxanyl [PROTECTIVEGROUPS IN ORGANIC SYNTHESIS, second edition, JOHN WILEY & SONS, INC.(1991)].

Examples of the polyols, polyhalides and polytosyls having a functionalgroup that can be utilized as R², as such or through introduction andremoval of a protective group, and being useful as a starting materialfor constructing the structure of the L moiety include shikimic acid,quinic acid, 3,4,5-trihydroxybenzoic acid, cholic acid, and halides andtosylates thereof.

Among Compounds (I), those obtained by introducing substituent R² intocompounds having L can readily be produced, for example, by thefollowing processes.

Process 1-1

Among Compounds (Ia),

those wherein R² is carboxy, i.e. compounds represented by formula(Iaa):(R¹—M_(n)—X^(1a))_(m)L (X²—X³-COOH)_(q)  (Iaa)(wherein X^(1a) represents a bond, O, alkylene, O(CH₂)_(ta) or(CH₂)_(tb)O; and R¹, L, M, n, m, q, X² and X³ have the same meanings asdefined above, respectively); those wherein R² is carbamoyl, i.e.compounds represented by formula (Iab):(R¹—M_(n)—X^(1a))_(m)L(X²—X³—CONH₂)_(q)  (Iab)(wherein R¹, L, M, n, m, q, X^(1a), X² and X³ have the same meanings asdefined above, respectively); and those wherein R² is cyano, i.e.compounds represented by formula (Iac):(R¹—M_(n)—X^(1a))_(m)L(X²—X³—CN)_(q)  (Iac)(wherein R¹, L, M, n, m, q, X^(1a), X² and X³ have the same meanings asdefined above, respectively) can be synthesized, for example, in thefollowing manner.

Compound (Iaa), Compound (Iab) and Compound (Iac) can be obtained byreacting a reaction mixture containing (Iaj), i.e. Compound (Ia) havinga hydroxyl group as R² among Compounds (Ia) obtained by Process 1 usinga polyol, or the compound purified from the mixture with 1 to 30 molarequivalents of acrylic acid, acrylamide, acrylonitrile or the like in anappropriate solvent (e.g. water, methylene chloride, toluene ortetrahydrofuran) in the presence of a base (catalytic amount or 1 to20%) at −20 to 150° C. for 1 hour to several days. Suitable basesinclude potassium hydroxide, sodium hydroxide and sodium hydride.Compound (Iaa) can also be obtained by dissolving or suspending areaction mixture containing Compound (Iaj) obtained by Process 1 or thecompound purified therefrom in an appropriate solvent (e.g.N,N-dimethylformamide, dimethyl sulfoxide, toluene or tetrahydrofuran)under anhydrous conditions, and reacting the compound with 1 to 50 molarequivalents of α-halogenated acetic acid ester in the presence of 1 to50 mol of an appropriate base (e.g. sodium hydride, zinc oxide, sodiumhydroxide or triethylamine) at −20 to 150° C. for 1 hour to severaldays, followed by hydrolysis. Further, Compound (Iaa) can be obtained bydissolving or suspending Compound (Iaj) obtained by Process 1 in anappropriate solvent (e.g. N,N-dimethylformamide, dimethyl sulfoxide,toluene or tetrahydrofuran), and reacting the compound with 1 to 50 molof an activating agent (e.g. succinimidyl carbonate, p-nitrophenylchloroformate or carbonyldiimidazole) in the presence of 1 to 50 mol ofan appropriate base (e.g. sodium hydride, zinc oxide, sodium hydroxideor triethylamine) at −20 to 100° C. for 1 hour to 10 days to activatethe compound, followed by reaction with an amino acid such asγ-aminobutyric acid, glycine or β-alanine, or a derivative thereof.

It is also possible to produce Compound (Iaa) by reacting Compound (Iaj)obtained by Process 1 with an acid anhydride such as succinic anhydrideor glutaric anhydride in the presence of the same base as above.

Moreover, Compound (Iaa) can be obtained by, after producing Compound(Iai), i.e. Compound (Ia) wherein R² is halogenated lower alkylaccording to Process 1 using a polyhalide, dissolving or suspendinghydroxycarboxylate, malonate, γ-aminobutyrate, an ester of β-alanine, anester of glycine or the like in an appropriate solvent (e.g.N,N-dimethylformamide, dimethyl sulfoxide, toluene or tetrahydrofuran),adding Compound (Iai) thereto in the presence of 1 to 50 mol of anappropriate base (e.g. sodium hydride, zinc oxide, sodium hydroxide ortriethylamine), and reacting them at −20 to 150° C. for 1 hour toseveral days, followed by hydrolysis.

Furthermore, Compound (Iaa) can be obtained by substituting at least onehydroxyl group or halogen of the above polyol or polyhalide with aresidue containing carboxylic acid or protected carboxylic acid inadvance, and then substituting the remaining three or more hydroxylgroups or halogens of the polyol or polyhalide with polyalkylene glycolA or a halide or tosylate thereof according to the method shown inProcess 1. In this case, the introduction of the residue containingcarboxylic acid or protected carboxylic acid can be carried out in amanner similar to the above. When carboxylic acid is protected, theprotective group is removed after the introduction of polyalkyleneglycol A or a halide or tosylate thereof into the polyol or polyhalideto form free carboxylic acid.

The compound converted into carboxylic acid can be purified and isolatedat a desired purity according to a known method such as anion-exchangechromatography, hydrophobic chromatography, reversed phasechromatography, two-phase partition or recrystallization.

Process 1-2

Among Compounds (Ia), those wherein R² is amino, i.e. compoundsrepresented by formula (Iad):(R¹—M_(n)—X^(1a))_(m)L(X²—X³—NH₂)_(q)  (Iad)(wherein R¹, L, M, n, m, q, X^(1a), X² and X³ have the same meanings asdefined above, respectively) can be obtained, for example, by treatingCompound (Iac) obtained by Process 1-1 with an appropriate reducingagent. Suitable reducing agents include lithium aluminum hydride, sodiumborohydride, sodium cyanoborohydride and hydrogen.

Compound (Iad) can also be obtained by reacting Compound (Iai) obtainedby Process 1 or a compound wherein the halogen moiety of Compound (Iai)is substituted with a tosyl group, with 5 equivalents to an excessamount of a diamine such as ethylenediamine or propylenediamine in thepresence of an appropriate base.

Further, similarly to Process 1-1, Compound (Iad) can be obtained bydissolving or suspending Compound (Iaj) in an appropriate solvent (e.g.N,N-dimethylformamide, dimethyl sulfoxide, toluene or tetrahydrofuran),and reacting the compound with 1 to 50 mol of an activating agent (e.g.succinimidyl carbonate, p-nitrophenyl chloroformate orcarbonyldiimidazole) in the presence of 1 to 50 mol of an appropriatebase (e.g. sodium hydride, zinc oxide, sodium hydroxide ortriethylamine) at −20 to 100° C. for 1 hour to 10 days to activate thecompound, followed by reaction with 1 equivalent to an excess amount ofa diamine such as ethylenediamine or propylenediamine in the presence ofan appropriate base.

Furthermore, Compound (Iad) can be obtained, in accordance with themethod shown in Process 1, by introducing at least one amino orprotected amino into a compound such as a polyol used for forming L inadvance, and then substituting the remaining three or more hydroxylgroups or halogen moieties of the compound with polyalkylene glycol A ora halide or tosylate thereof.

Among Compounds (Ia), those wherein R² is maleimido, i.e. compoundsrepresented by formula (Iae):

(wherein R¹, L, M, n, m, q, X^(1a), X² and X³ have the same meanings asdefined above, respectively) can be obtained, for example, by reactingCompound (Iad) with N-alkoxycarbonylmaleimide in a saturated aqueoussolution of sodium hydrogencarbonate according to the method of OskarKeller, et al. [Helv. Chim. Acta, Vol. 58, p. 531 (1975)] or the methodof Timothy P. Kogan, et al. [Synthetic Commun., Vol. 22, p. 2417(1992)]. As the N-alkoxycarbonylmaleimide, N-ethoxycarbonylmaleimide andN-methoxycarbonylmaleimide can be used.

Compound (Iae) can also be obtained, in accordance with the method shownin Process 1, by introducing at least one maleimido into a compound suchas a polyol used for forming L in advance, and then substituting theremaining three or more hydroxyl groups or halogen moieties of thecompound with polyalkylene glycol A or a halide or tosylate thereof.

Compound (Iad), Compound (Iae) and synthetic intermediates thereof canbe isolated and purified to a desired purity according to the number ofbranches of polyalkylene glycol by the same methods as above.

Process 1-3

Among Compounds (Ia), those wherein R² is formyl, i.e. compoundsrepresented by formula (Iaf):(R¹—M_(n)—X^(1a))_(m)L(X²—X³C(═O)H)_(q)  (Iaf)(wherein R¹, L, M, n, m, q, X^(1a), X² and X³ have the same meanings asdefined above, respectively) can be obtained, for example, by oxidizingCompound (Iag), i.e. Compound (Ia) having hydroxylmethyl as R² obtainedby Process 1 with an appropriate oxidizing agent. Suitable oxidizingagents include pyridinium chlorochromate, chromic acid, silver ion anddimethyl sulfoxide. Compound (Iaf) can also be obtained by reducingCompound (Iaa) with an appropriate reducing agent in a manner similar tothe above.

Moreover, formyl can be introduced by binding aminoethyl acetal,hydroxyethyl acetal, halogenated ethyl acetal, halogenated methyl acetalor the like to Compound (Iaj) or Compound (Iai) obtained by Process 1 ora compound wherein the halogen moiety of Compound (Iai) is substitutedwith a tosyl group, and then removing acetal.

Similarly, using Compound (Iaj) obtained by Process 1, formyl can alsobe introduced by activating a hydroxyl group according to the methodshown in Process 1-1, binding aminoethyl acetal, hydroxyethyl acetal orthe like, and then removing acetal.

Compound (Iaf) can also be obtained, in accordance with the method shownin Process 1, by introducing at least one aldehyde or protected aldehydeinto a compound such as a polyol used for forming L in advance, and thensubstituting the remaining three or more hydroxyl groups or halogenmoieties of the compound with polyalkylene glycol A or a halide ortosylate thereof.

Compound (Iaf) and synthetic intermediates thereof can be isolated andpurified to a desired purity according to the number of branches ofpolyalkylene glycol by the same methods as above.

Process 1-4

Among Compounds (Ia), those wherein R² is halogenated carbonyl, i.e.compounds represented by formula (Iah):(R¹—M_(n)—X^(1a))_(m)L(X²—X³—C(═O) Z¹)_(q)  (Iah)(wherein Z¹ represents a halogen; and R¹, L, M, n, m, q, X^(1a), X² andX³ have the same meanings as defined above, respectively) can beobtained, for example, by heating Compound (Iaa) having carboxy as R² inthionyl halide or in an appropriate mixed solvent of thionyl halide andtoluene, dimethylformamide or the like in the presence of an appropriatecatalyst (e.g. pyridine or triethylamine) at 0 to 150° C. for 1 to 24hours.

The halogen in the halogenated carbonyl has the same meaning as theabove halogen.

Process 1-5

Among Compounds (Ia), those wherein R² is halogenated lower alkyl, i.e.compounds represented by formula (Iai):(R¹—M_(n)—X^(1a))_(m)L(X²—X³—Z²)_(q)  (Iai)(wherein Z² represents a halogenated lower alkyl; and R¹, L, M, n, m, q,X^(1a), X² and X³ have the same meanings as defined above, respectively)can be obtained, for example, by heating Compound (Iaj) having ahydroxyl group as R² in thionyl halide or in an appropriate mixedsolvent of thionyl halide and toluene, dimethylformamide or the like inthe presence of an appropriate catalyst (e.g. pyridine or triethylamine)at 0 to 150° C. for 1 to 24 hours.

The halogen and the lower alkyl moiety in the halogenated lower alkylhave the same meanings as defined above, respectively.

Compound (Iai) can also be obtained by reacting Compound (Iaj) obtainedby Process 1 or Compound (Iad) having amino as R² with 5 equivalents toan excess amount of dihalogenated alkyl such as dibromoethane ordibromopropane in the presence of an appropriate base as describedabove.

Further, Compound (Iai) can be obtained, in accordance with the methodshown in Process 1 above, by introducing at least one halogenated loweralkyl into a compound such as a polyol used for forming L in advance,and then substituting the remaining three or more hydroxyl groups orhalogen moieties of the compound with polyalkylene glycol A or a halideor tosylate thereof.

Compound (Iai) and synthetic intermediates thereof can be isolated andpurified to a desired purity according to the number of branches ofpolyalkylene glycol by the same methods as above.

Process 1-6

Among Compounds (Ia), those wherein R² is isocyanato, i.e. compoundsrepresented by formula (Iak):(R¹—M_(n)—X^(1a))_(m)L(X²—X³—N═C═O)_(q)  (Iak)(wherein R¹, L, M, n, m, q, X^(1a), X² and X³ have the same meanings asdefined above, respectively) can be obtained, for example, by reactingCompound (Iad) with phosgene or oxalyl chloride in an appropriatesolvent (e.g. toluene, tetrahydrofuran or methylene chloride) at 0 to150° C. for 1 to 24 hours, or by reacting the compound withN,N′-carbonyldiimidazole, followed by decomposition at room temperature.

Compound (Iap), i.e. Compound (Ia) wherein R² is isothiocyanato (—N═C═S)can be produced according to the same process as above except thatthiophosgene is used in place of phosgene.

Process 1-7

Among Compounds (Ia), those wherein R² is succinimidooxycarbonyl,substituted or unsubstituted aryloxycarbonyl, benzotriazolyloxycarbonylor phthalimidooxycarbonyl, i.e. compounds represented by formula (Ial):(R¹—M_(n)—X^(1a))_(m)L(X²—X³—R^(2a))_(q)  (Ial)(wherein R^(2a) represents succinimidooxycarbonyl, substituted orunsubstituted aryloxycarbonyl, benzotriazolyloxycarbonyl orphthalimidooxycarbonyl; and R¹, L, M, n, m, q, X^(1a), X² and X³ havethe same meanings as defined above, respectively) can be produced byordinary methods for synthesizing esters.

For example, the desired compound can be obtained by reacting 1 mol ofCompound (Iaa) with 1 to 10 mol of N-hydroxysuccinimide, substituted orunsubstituted hydroxyaryl, N-hydroxybenzotriazole orN-hydroxyphthalimide in the presence of 1 to 10 mol of a condensingagent (e.g. N,N′-dicyclohexylcarbodiimide) in an appropriate solvent(e.g. dimethylformamide, methylene chloride or dimethyl sulfoxide) at−20 to 100° C. for 1 to 24 hours. More specifically, the desiredcompound can be obtained according to the method of introducing acarboxyl group to an end of polyalkylene glycol, the method of producingN-hydroxysuccinimide ester of carboxymethylpolyalkylene glycol, or thelike by A. Fradet, et al. [Polym. Bull., Vol. 4, p. 205 (1981)] or K.Geckeler, et al. [Polym. Bull., Vol. 1, p. 691 (1979)].

The substituted or unsubstituted aryloxycarbonyl has the same meaning asdefined above. The aryl moiety of the hydroxyaryl has the same meaningas the aryl moiety of the aryloxycarbonyl, and the substituent in thesubstituted hydroxyaryl has the same meaning as the substituent in thesubstituted aryloxycarbonyl.

Process 1-8

Among Compounds (Ia), those wherein R² is vinylsulfonyl, i.e. compoundsrepresented by formula (Iam):(R¹—M_(n)—X^(1a))_(m)L(X²—X³—SO₂—CH═CH₂)_(q)  (Iam)(wherein R¹, L, M, n, m, q, X^(1a), X² and X³ have the same meanings asdefined above, respectively) can be produced, for example, by the methodof Margherita Morpurgo, et al. [Bioconjugate Chem., Vol. 7, p. 363(1996)] using Compound (Iaj).Process 1-9

Among Compounds (Ia), those wherein R² is substituted or unsubstitutedlower alkoxycarbonyloxy or substituted or unsubstitutedaryloxycarbonyloxy, i.e. compounds represented by formula (Ian):(R¹—M_(n)—X_(1a))_(m)L(X²—X³—R^(2b))  (Ian)(wherein R^(2b) represents substituted or unsubstituted loweralkoxycarbonyloxy or substituted or unsubstituted aryloxycarbonyloxy;and R¹, L, M, n, m, q, X^(1a), X² and X³ have the same meanings asdefined above, respectively) can be obtained, for example, by reactingCompound (Iaj) having a hydroxyl group as R² with an excess amount ofp-nitrophenyl chloroformate, ethyl chloroformate or the like in thepresence of a base (e.g. dimethylaminopyridine or triethylamine)according to the method of Talia Miron and Meir Wilchek [BioconjugateChem., Vol. 4, p. 568 (1993)].

Compound (Ian) can also be obtained, in accordance with the method shownin Process 1, by introducing at least one substituted or unsubstitutedalkoxycarbonyloxy or substituted or unsubstituted aryloxycarbonyloxyinto a compound such as a polyol used for forming L in advance, and thensubstituting the remaining three or more hydroxyl groups or halogenmoieties of the compound with polyalkylene glycol A or a halide ortosylate thereof.

Compound (Ian) and synthetic intermediates thereof can be isolated andpurified to a desired purity according to the number of branches ofpolyalkylene glycol by the same methods as above.

The substituted or unsubstituted lower alkoxycarbonyloxy and thesubstituted or unsubstituted aryloxycarbonyloxy have the same meaningsas defined above, respectively.

Process 2: Compounds wherein X¹ is S

Compound (Ib), i.e. Compound (I) wherein X¹ is S can be obtained, forexample, in a manner similar to that in Process 1, by reacting acompound obtained by converting a polyol into a polyhalide [edited byThe Chemical Society of Japan, Jikken Kagaku Koza (ExperimentalChemistry Course), fourth edition, Vol. 19 (1992), Maruzen] or acommercially available polyhalide with a thiol derivative ofpolyalkylene glycol A in an appropriate solvent in the presence of anappropriate base.

Compound (Ib) can also be obtained, in reverse to the above step, byreacting a halide or tosylate of polyalkylene glycol A with a polythiol.

The thiol derivative of polyalkylene glycol A may be a commerciallyavailable product or may be prepared by the methods summarized by SamuelZalipsky, et al. [Bioconjugate Chem., Vol. 6, p. 150 (1995)].

The reaction conditions and purification conditions in each step aresimilar to those in Process 1.

Process 2-1

Among Compounds (Ib), those wherein R² is carboxy, carbamoyl, cyano,amino, maleimido, formyl, halogenated carbonyl, halogenated lower alkyl,isocyanato, isothiocyanato, succinimidooxycarbonyl, substituted orunsubstituted aryloxycarbonyl, benzotriazolyloxycarbonyl,phthalimidooxycarbonyl, vinylsulfonyl, substituted or unsubstitutedlower alkoxycarbonyloxy, or substituted or unsubstitutedaryloxycarbonyloxy can be obtained by producing the compound wherein X¹is —S— according to Process 2, and then combining the methods describedin Process 1-1 to Process 1-9.

Process 3: Compounds wherein X¹ is NR³

Compound (Ic), i.e. Compound (I) wherein X¹ is NR³ (in which R³ has thesame meaning as defined above) can be obtained, for example, in a mannersimilar to that in Process 1, by reacting a compound obtained byconverting a polyol into a polyamine or a commercially availablepolyamine with a halide or tosylate of polyalkylene glycol A in anappropriate solvent in the presence of an appropriate base.

Compound (Ic) can also be obtained by reacting an amino derivative ofpolyalkylene glycol A with a polyhalide.

Moreover, Compound (Ic) can be obtained by dissolving or suspending apolyaldehyde (1 equivalent) and an amino derivative of polyalkyleneglycol A (1 to 30 equivalents per formyl group in the polyaldehyde) inan appropriate solvent (e.g. methanol, ethanol, dimethylformamide,acetonitrile, dimethyl sulfoxide, water or buffer), and reacting them inthe presence of a reducing agent (e.g. sodium cyanoborohydride or sodiumborohydride; 1 to 30 equivalents per formyl group in the polyaldehyde)at −20 to 100° C.

Further, Compound (Ic) can be produced by using a polyamine and analdehyde derivative of polyalkylene glycol A.

As the above polyaldehyde, a commercially available one may be used asit is. Also useful are a compound obtained by oxidizing a polyalcohol,and a compound obtained by reducing a polycarboxylic acid. The aldehydederivative of polyalkylene glycol A may be a commercially availableproduct, or may be prepared by oxidizing alcohol at an end ofpolyalkylene glycol A.

The reaction conditions and purification conditions in each step aresimilar to those in Process 1.

Process 3-1

Among Compounds (Ic), those wherein R² is carboxy, carbamoyl, cyano,amino, maleimido, formyl, halogenated carbonyl, halogenated lower alkyl,isocyanato, isothiocyanato, succinimidooxycarbonyl, substituted orunsubstituted aryloxycarbonyl, benzotriazolyloxycarbonyl,phthalimidooxycarbonyl, vinylsulfonyl, substituted or unsubstitutedlower alkoxycarbonyloxy, or substituted or unsubstitutedaryloxycarbonyloxy can be obtained by synthesizing Compound (Ic)according to Process 3, and then combining the methods described inProcess 1-1 to Process 1-9.

Process 4: Compounds wherein X¹ is R⁴—NH—C(═O)—R⁵ or R⁶—C(═O)—NH—R⁷

Compound (Ida), i.e. Compound (I) wherein X¹ is R⁴—NH—C(═O)—R⁵ (in whichR⁴ and R⁵ have the same meanings as defined above, respectively) can beobtained, for example, by dissolving or suspending a polycarboxylic acidcompound selected from γ-carboxyglutamic acid, citric acid,1,2,3,4-butanetetracarboxylic acid, etc. in an appropriate solvent (e.g.N,N-dimethylformamide or dimethyl sulfoxide), adding an alcohol compound(e.g. N-hydroxysuccinimide, N-hydroxyphthalimide, N-hydroxybenzotriazoleor p-nitrophenol; 1 to 30 equivalents per carboxyl group in thepolycarboxylic acid compound) and a condensing agent (e.g.N,N′-dicyclohexylcarbodiimide orbenzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate; 1 to30 equivalents per carboxyl group in the polycarboxylic acid compound),further adding an amino derivative of polyalkylene glycol A (1 to 30equivalents per carboxyl group in the polycarboxylic acid compound), andreacting them according to a peptide synthetic method [Izumiya, et al.,Peptide Gosei no Kiso to Jikken (Basis and Experiment of PeptideSynthesis) (1985), Maruzen]. The reaction is carried out with stirringunder anhydrous conditions at −20 to 100° C. for 1 hour to 10 days.

It is also possible to obtain a reaction mixture containing a branchedpolyethylene glycol derivative having three or more chains wherein R² iscarboxy at a high purity by protecting at least one carboxyl group in apolycarboxylic acid molecule with an appropriate protective group (e.g.methyl, ethyl, benzyl or tert-butyl), introducing an amino derivative ofpolyalkylene glycol A to the remaining carboxyl groups by the abovemethod, and then removing the protective group of the carboxyl group bya usual deprotection method. In this case, the introduction and removalof the protective group of carboxylic acid can be carried out by usingmethods employed in ordinary peptide synthesis [Izumiya, et al., PeptideGosei no Kiso to Jikken (Basis and Experiment of Peptide Synthesis)(1985), Maruzen]. The configuration of carboxyl groups in thepolycarboxylic acid may be any configuration including stericconfiguration. The amino derivative of polyalkylene glycol A used abovemay have any average molecular weight so long as the molecular weightdistribution is uniform (preferably Mw/Mn is 1.1 or less).

Compound (Idb), i.e. Compound (I) wherein X¹ is R⁶—C(═O)—NH—R⁷ (in whichR⁶ and R⁷ have the same meanings as defined above, respectively) canalso be obtained, in reverse to the above step, by reacting a polyaminewith an active ester of a carboxylic acid derivative of polyalkyleneglycol A or an acid halide derivative of polyalkylene glycol A. The acidhalide derivative of polyalkylene glycol A can be obtained by heating acarboxylic acid derivative of polyalkylene glycol A in thionyl halide orin an appropriate mixed solvent of thionyl halide and toluene,dimethylformamide or the like in the presence of an appropriate catalyst(e.g. pyridine or triethylamine) at 0 to 150° C. for 1 to 24 hours.

The reaction conditions and purification conditions in each step aresimilar to those in the above processes.

Process 4-1

Among Compounds (Ida) and (Idb), those wherein R² is carboxy, carbamoyl,cyano, amino, maleimido, formyl, halogenated carbonyl, halogenated loweralkyl, isocyanato, isothiocyanato, succinimidooxycarbonyl, substitutedor unsubstituted aryloxycarbonyl, benzotriazolyloxycarbonyl,phthalimidooxycarbonyl, vinylsulfonyl, substituted or unsubstitutedlower alkoxycarbonyloxy, or substituted or unsubstitutedaryloxycarbonyloxy can be obtained by synthesizing Compound (Ida) orCompound (Idb) according to Process 4, and then combining the methodsdescribed in Process 1-1 to Process 1-9.

Process 5: Compounds wherein X¹ is R⁸—C(═O)—O or O—C(═O)—R⁹

Compound (Ie), i.e. Compound (I) wherein X¹ is R⁸—C(═O)—O (in which R⁸has the same meaning as defined above) or O—C(═O)—R⁹ (in which R⁹ hasthe same meaning as defined above) can be obtained, for example, bydehydration condensation using a combination of polyalkylene glycol Aand a polycarboxylic acid, or a carboxylic acid derivative ofpolyalkylene glycol A and a polyol. Dehydration condensation can becarried out by dehydration in the presence of an acid or base catalystas in ordinary ester synthesis, or by condensing a corresponding alcoholcompound and carboxylic acid using a condensing agent such asN,N′-dicyclohexylcarbodiimide in an appropriate solvent (e.g.dimethylformamide, dimethyl sulfoxide, acetonitrile, pyridine ormethylene chloride). The desired compound can also be synthesized byreacting an acid halide with a corresponding alcohol compound in theabove step.

The reaction conditions and purification conditions in each step aresimilar to those in the above processes.

Process 5-1

Among Compounds (Ie), those wherein R² is carboxy, carbamoyl, cyano,amino, maleimido, formyl, halogenated carbonyl, halogenated lower alkyl,isocyanato, isothiocyanato, succinimidooxycarbonyl, substituted orunsubstituted aryloxycarbonyl, benzotriazolyloxycarbonyl,phthalimidooxycarbonyl, vinylsulfonyl, substituted or unsubstitutedlower alkoxycarbonyloxy, or substituted or unsubstitutedaryloxycarbonyloxy can be obtained by synthesizing Compound (Ie)according to Process 5, and then combining the methods described inProcess 1-1 to Process 1-9.

Process 6: Compounds wherein X¹ is R^(6a)—O—C(═O)—NH or R⁴—NH—C(═O)—O

Compound (Ifa), i.e. Compound (I) wherein X¹ is R^(6a)—O—C(═O)—NH (inwhich R^(6a) has the same meaning as defined above) can be produced, forexample, in the following manner.

A crude product containing Compound (Ifa) can be obtained by reacting acommercially available polyamine or a polyamine prepared from a polyolby a combination of the above processes with at least 3 mol of acarbonate derivative of polyalkylene glycol A. The carbonate derivativeof polyalkylene glycol A can be produced according to the method ofTalia Miron, et al. [Bioconjugate Chem., Vol. 4, p. 568 (1993)]. As thecarbonate derivative of polyalkylene glycol A, N-hydroxysuccinimidylcarbonate, p-nitrophenyl carbonate, imidazolylcarbonyloxy derivative,etc. can be used.

Compound (Ifb), i.e. Compound (I) wherein X¹ is R⁴—NH—C(═O)—O (in whichR⁴ has the same meaning as defined above) can be produced, for example,in the following manner.

Compound (Ifb) can be obtained by reacting a carbonate derivative of apolyol with an amino derivative of polyalkylene glycol A in a mannersimilar to the above.

It is also possible to selectively form Compound (Ifa) or Compound (Ifb)by combining protection and deprotection of a functional group accordingto other processes.

The reaction conditions and purification conditions in each step aresimilar to those in the above processes.

Process 6-1

Among Compounds (If), those wherein R² is carboxy, carbamoyl, cyano,amino, maleimido, formyl, halogenated carbonyl, halogenated lower alkyl,isocyanato, isothiocyanato, succinimidooxycarbonyl, substituted orunsubstituted aryloxycarbonyl, benzotriazolyloxycarbonyl,phthalimidooxycarbonyl, vinylsulfonyl, substituted or unsubstitutedlower alkoxycarbonyloxy, or substituted or unsubstitutedaryloxycarbonyloxy can be prepared by synthesizing Compound (If)according to Process 6, and then combining the methods described inProcess 1-1 to Process 1-9.

It is also possible to obtain a single- or double-chain compound bybinding R¹—M_(n)—X¹ to L, and then obtain a compound having three ormore chains by binding R¹—M_(n)—X¹ which is the same or different fromthe above to L through similar reaction. For example, a single- ordouble-chain compound is obtained by binding polyalkylene glycol to oneor two functional groups in L by utilizing reaction selected from thoseshown in Processes 1 to 6. The content of the single- or double-chaincompound formed can be controlled by changing the ratio of thepolyalkylene glycol used in the reaction to the starting material forconstructing the structure of L moiety, and thus it is possible toproduce the single- or double-chain compound as a main component. Theobtained single- or double-chain compound can be used in the next stepat the purity as it is or after purifying it to a desired purityaccording to the number of branches of polyalkylene glycol or to a highpurity by the method shown in Process 1.

A compound having three or more chains can be prepared by bindingpolyalkylene glycol which is the same or different from the above to thesingle- or double-chain compound obtained above according to the methodselected from those shown in Processes 1 to 6. The third or furtherpolyalkylene glycol may be subjected to reaction similar to that forobtaining the single- or double-chain compound, or may be subjected to adifferent reaction so as to have a different binding mode. For example,when a compound having two or more functional groups such as a hydroxylgroup, amino and carboxy is used as a starting material for constructingthe structure of L moiety, it is possible to first obtain a single- ordouble-chain compound wherein X¹ is O by the method shown in Process 1,and then subject the third or further polyalkylene glycol to reaction sothat X¹ becomes R⁴—NH—C(═O)—R⁵ by the method shown in Process 4. Asdescribed above, a compound having three or more chains wherein pluralpolyalkylene glycols are bound to L in the same or different bindingmode can be obtained by combining Processes 1 to 6. The molecularweights of polyalkylene glycols used in the respective reaction stepsmay be different, and a desired compound can readily be obtained byusing polyalkylene glycols having different average molecular weights inthe respective reactions for binding polyalkylene glycols to L.

In the reaction for introducing polyalkylene glycols to L, it is alsopossible to protect functional groups in L with appropriate protectivegroups with the exception of at least one functional group (e.g. inProcess 1, at least one hydroxyl group) left unprotected, allow L toreact with polyalkylene glycols for binding, and then remove theprotective groups.

The branched polyalkylene glycols of the present invention other thanthe compounds specifically shown in the above processes can also beobtained according to processes similar to those described above.

As described above, the polyalkylene glycols used as starting materialsin Processes 1 to 6 are commercially available, but can also be easilyproduced by various methods summarized by Samuel Zalipsky [BioconjugateChem., Vol. 6, p. 150 (1995)], etc.

The obtained branched polyalkylene glycols can be purified to a desiredpurity according to the number of branches by methods such as silica gelchromatography, reversed phase chromatography, hydrophobicchromatography, ion-exchange chromatography, gel filtrationchromatography, recrystallization and extraction.

The resulting branched polyalkylene glycols can be bound to an aminoacid side chain, the N-terminal amino group or the C-terminal carboxylgroup of the above physiologically active polypeptide directly orthrough a spacer.

As the spacer, amino acids and peptides are preferably used, but othersubstances may also be used so long as they can bind to polyalkyleneglycols. Suitable amino acids include natural amino acids such as lysineand cysteine, as well as ornithine, diaminopropionic acid, homocysteineand the like. Preferred is cysteine. Preferred peptides are thoseconsisting of 2 to 10 amino acid residues. The spacers other than aminoacids and peptides include glycerol, ethylene glycol and sugars.Suitable sugars include monosaccharides and disaccharides such asglucose, galactose, sorbose, galactosamine and lactose.

The spacer is bound to a side chain of the residue of lysine, cysteine,arginine, histidine, serine, threonine or the like in a physiologicallyactive polypeptide molecule through an amide bond, a thioether bond, anester bond, etc., to the C-terminal carboxyl group of the polypeptidethrough an amide bond or an ester bond, or to the N-terminal amino groupof the polypeptide through an amide bond. The binding can be effected byordinary peptide synthetic methods [Izumiya, et al., Peptide Gosei noKiso to Jikken (Basis and Experiment of Peptide Synthesis) (1985),Maruzen] or recombinant DNA techniques.

It is preferred to introduce an amino acid, a peptide or the like as aspacer to the C-terminal carboxyl group of a physiologically activepolypeptide simultaneously with the synthesis of the physiologicallyactive polypeptide, but the spacer may be bound after the synthesis ofthe physiologically active polypeptide. It is also possible to activatethe C-terminal carboxyl group or the like of the polypeptide in achemical synthetic manner and then bind it to the spacer. Further, aspacer bound to polyalkylene glycol in advance may be bound to aphysiologically active polypeptide by the method described above.

The physiologically active polypeptides used in the present inventioninclude polypeptides, antibodies, and derivatives thereof. Examples ofthe polypeptides include enzymes such as asparaginase, glutaminase,arginase, uricase, superoxide dismutase, lactoferin, streptokinase,plasmin, adenosine deaminase, plasminogen activator and plasminogen;cytokines such as interleukin-1 to 18, interferon-α, interferon-β,interferon-γ, interferon-ω, interferon-τ, granulocyte-colony stimulatingfactor, thrombopoietin, erythropoietin, tumor necrosis factor,fibroblast growth factor-1 to 18, midkine, epidermal growth factor,osteogenic protein 1, stem cell factor, vascular endothelial growthfactor, transforming growth factor and hepatocyte growth factor;hormones such as glucagon, parathyroid hormone and glucagon likepeptide; klotho protein, angiopoietin, angiostatin, leptin, calcitonin,amylin, insulin like growth factor 1 and endostatin.

The antibodies used in the present invention can be obtained aspolyclonal antibodies or monoclonal antibodies by using a known method[Antibodies—A Laboratory Manual, Cold Spring Harbor Laboratory (1988)].

The antibody used in the present invention may be either a polyclonalantibody or a monoclonal antibody, but a monoclonal antibody ispreferred.

The monoclonal antibodies of the present invention include antibodiesproduced by hybridomas, humanized antibodies, and fragments thereof.

The humanized antibodies include human chimera antibodies and humanCDR-grafted antibodies.

By “human chimera antibodies” is meant antibodies comprising theheavy-chain variable region (hereinafter, also referred to as HV or VH,the heavy chain being referred to as H chain and the variable region asV region) and the light-chain variable region (hereinafter, alsoreferred to as LV or VL, the light chain being referred to as L chain)of an antibody derived from a non-human animal, and the heavy-chainconstant region (hereinafter, also referred to as CH, the constantregion being referred to as C region) and the light-chain constantregion (hereinafter, also referred to as CL) of a human antibody. As thenon-human animal, any animal can be used so far as hybridomas can beprepared from the animal. Suitable animals include mouse, rat, hamsterand rabbit.

By “human CDR-grafted antibodies” is meant antibodies prepared bygrafting the amino acid sequences of the CDR in the V regions of H chainand L chain of an antibody of a non-human animal into appropriate sitesin the V regions of H chain and L chain of a human antibody.

The antibody fragments include Fab, Fab′, F(ab′)₂, single-chainantibodies, disulfide-stabilized V region fragments, and peptidescomprising a complementarity determining region.

Fab is a fragment with a molecular weight of about 50,000 havingantigen-binding activity constituted of about half of H chain(N-terminal side) and the full L chain, which is obtained by cleavingthe peptide moiety above two disulfide bonds cross-linking two H chainsin the hinge regions of IgG with papain.

Fab′ is a fragment with a molecular weight of about 50,000 havingantigen-binding activity, which is obtained by cleaving disulfide bondsof the hinge regions of the above F(ab′)₂.

F(ab′)₂ is a fragment with a molecular weight of about 100,000 havingantigen-binding activity constituted of two Fab regions bound at thehinge regions, which is obtained by cleaving the part below twodisulfide bonds in the hinge regions of IgG with trypsin.

The single-chain antibody (hereinafter also referred to as scFv) refersto a VH-P-VL or VL-P-VH polypeptide in which one VH and one VL arelinked using an appropriate peptide linker (hereinafter referred to asP). The VH and VL contained in the scFv used in the present inventionmay be any of the monoclonal antibody and the human CDR-grafted antibodyof the present invention.

The disulfide-stabilized V region fragment (hereinafter also referred toas dsFv) is a fragment in which polypeptides prepared by substitutingone amino acid residue in each of VH and VL with a cysteine residue arelinked via a disulfide bond. The amino acid residue to be substitutedwith a cysteine residue can be selected based on the prediction of thethree-dimensional structure of antibody according to the method shown byReiter, et al. [Protein Engineering, Vol. 7, p. 697 (1994)]. The VH andVL contained in the disulfide-stabilized antibody of the presentinvention may be any of the monoclonal antibody and the humanCDR-grafted antibody.

The derivatives of the physiologically active polypeptides include aminoacid-substituted derivatives, amino acid-deleted derivatives, sugarchain-added derivatives, sugar chain-deleted derivatives and partialpeptides.

Among the physiologically active polypeptides and derivatives thereofdescribed above, preferred examples include interferons such asinterferon-β, interferon-α and interferon-γ, granulocyte-colonystimulating factor and superoxide dismutase.

These physiologically active polypeptides can be obtained not only byextraction from animal organs and tissues, but also by ordinary peptidesynthesis and recombinant DNA techniques. Commercially availablepolypeptides can also be used.

The polypeptide used in the reaction may be a partially purified productor a product purified to a purity suitable for chemical modification bypurification methods such as gel filtration chromatography, ion-exchangechromatography, hydrophobic chromatography, reversed phasechromatography and extraction.

The polypeptide is produced in a buffer such as a phosphate buffer, aborate buffer, an acetate buffer or a citrate buffer, water, anappropriate organic solvent such as N,N-dimethylformamide, dimethylsulfoxide, dioxane or tetrahydrofuran, or a mixed solvent of such anorganic solvent and an aqueous solution, and then used in chemicalmodification reaction.

The branched polyalkylene glycols of the present invention can also beused for site-specific covalent modification of polypeptides, morespecifically and preferably, all natural or recombinant polypeptideshaving a free cysteine residue such as granulocyte-colony stimulatingfactor (G-CSF), erythropoietin, interferons and interleukins.

The physiologically active polypeptide modified with the branchedpolyalkylene glycol of the present invention is produced by reactionusing the branched polyalkylene glycol in an amount of 1 to 1000 mol,preferably 1 to 50 mol per mol of a physiologically active polypeptide.The degree of modification of the physiologically active polypeptidewith the branched polyalkylene glycol can be arbitrarily selected bycontrolling the molar ratio of the branched polyalkylene glycol to thephysiologically active polypeptide, reaction temperature, pH, reactiontime, etc. The solvent used in the reaction may be any of the solventsthat do not interfere with the reaction, for example, a phosphatebuffer, a borate buffer, a tris-hydrochloride buffer, an aqueous sodiumhydrogencarbonate solution, a sodium acetate buffer,N,N-dimethylformamide, dimethyl sulfoxide, methanol, acetonitrile anddioxane. The temperature, pH and time of the reaction are not limited solong as the activity of the physiologically active polypeptide is notimpaired under the conditions. For example, the reaction is preferablycarried out at 0 to 50° C. at pH 4 to 10 for 10 minutes to 100 hours.

The physiologically active polypeptide modified with the branchedpolyalkylene glycol of the present invention can be purified by gelfiltration, ion-exchange chromatography, reversed phase high performanceliquid chromatography, affinity chromatography, ultrafiltration or thelike in a usual manner. Confirmation of the polypeptide structure in thesynthesized or purified physiologically active polypeptide or thephysiologically active polypeptide modified with the branchedpolyalkylene glycol can be carried out by mass spectrometry, nuclearmagnetic resonance (NMR) and amino acid composition analysis using anamino acid analyzer, and also by amino acid sequence analysis by use ofa gas phase protein sequencer in which phenylthiohydantoin (PTH) aminoacid obtained by Edman degradation is analyzed by reversed phase HPLC.

The chemically modified polypeptide of the present invention can beadministered in the form of a pharmaceutical composition for human oranimals, and the composition can be produced by ordinary methods forproducing pharmaceuticals. The methods of administration include oral,intravenous, subcutaneous, submuscular, intraperitoneal and percutaneousadministration and other acceptable methods, and a composition suitablefor administration can be used. The composition may comprise generallyemployed additives such as an isotonizing agent, a buffer, an excipient,a pH regulator, a stalilizer, an antiseptic, a solubilizing agent, awetting agent, an emulsifier, a lubricant, a sweetener, a coloring agentand an antioxidant.

Specific examples of Compounds (I) are shown in Tables 1 and 2.

The following are supplementary explanations of the structure of thecompounds shown in Table 1.

-   1) In Compound 5TRC(3UA) obtained in Example 1, the carboxyl group    corresponding to (X²—X³-R²) binds to the methylene group of —NHCH₂—.    CH₃—(OCH₂CH₂)_(n)—NH(C═O)—corresponding to [CH₃—(OCH₂CH₂)_(n)—X¹]    binds to the methyleneoxy groups (—CH₂O—).-   2) In Compound 5SKA(3UA) obtained in Example 2, the carboxyl group    corresponding to (X²—X³-R²) binds to the 1-position of the    cyclohexene ring. CH₃—(OCH₂CH₂)_(n)—NH(C═O)—corresponding to    [CH₃—(OCH₂CH₂)_(n)-X¹] binds to the oxygen atoms at the 3-, 4- and    5-positions of the cyclohexene ring.-   3) In Compound 5QNA(4UA) obtained in Example 3, the carboxyl group    corresponding to (X²—X³—R²) binds to the 1-position of the    cyclohexane ring, and the carboxyl group sterically exists in the    upward direction from the plane of the figure.    CH₃—(OCH₂CH₂)_(n)—NH(C═O)—O— binding to the 1-position sterically    exists in the downward direction from the plane of the figure.    CH₃—(OCH₂CH₂)—NH(C═O)—corresponding to [CH₃—(OCH₂CH₂)_(n)—X¹] binds    to the oxygen atoms at the 1-, 3-, 4- and 5-positions of the    cyclohexane ring.-   4) In Compound 5PET(3UA) obtained in Example 4,    —O—(C═O)—NH(CH₂)₃COOH corresponding to (X²—X³—R²) binds to the    methylene group (—CH₂—). CH₃—    (OCH₂CH₂)_(n)—CH₂—NH(C═O)—corresponding to [CH₃—(OCH₂CH₂)—X¹] binds    to the methyleneoxy groups (—CH₂O—).-   5) In Compound 5PET(3UM) obtained in Example 5, the    3-maleimidopropylaminocarbonyloxy group corresponding to (X²—X³—R²)    binds to the methylene group (—CH₂—). CH₃—(OCH₂CH₂)_(n)—CH₂—NH(C═O)—    corresponding to [CH₃—(OCH₂CH₂)_(n)—X¹] binds to the methyleneoxy    groups (—CH₂O—).-   6) In Compound 5PET(3UU) obtained in Example 6, the    maleimidooxycarbonyloxy group corresponding to (X²—X³—R²) binds to    the methylene group (—CH₂—). CH₃—(OCH₂CH₂)_(n)—CH₂—NH(C═O)—    corresponding to [CH₃—(OCH₂CH₂)_(n)-X¹] binds to the methyleneoxy    groups (—CH₂O—).-   7) In Compound 5PET(3URa) obtained in Example 7,    —O—(C═O)—NH(CH₂)₃CHO corresponding to (X²—X³-R²) binds to the    methylene group (—CH₂—). CH₃—(OCH₂CH₂)_(n)—CH₂—NH(C═O)—corresponding    to [CH₃—(OCH₂CH₂)_(n)-X¹] binds to the methyleneoxy groups (—CH₂O—).

8) In Compound 5SUG(4UA) obtained in Example 8, the carboxyl group—O—(C═O) corresponding to (X²—X³-R²) binds to the oxymethylene group(—OCH₂—) at the 1-position. CH₃—(OCH₂CH₂)_(n)—CH₂—NH(C═O)— correspondingto [CH₃—(OCH₂CH₂)_(n)—X¹] binds to the oxygen atoms at the 2-, 3- and4-positions and the methyleneoxy group at the 5-position. TABLE 1[CH₃—(OCH₂CH₂)_(n)—X¹]_(m)L—(X²—X³—R²)_(q) (I) Example No. Abbrev. X¹ mL q X²—X³—R² 1 5TRC(3UA)

3

1

2 5SKA(3UA)

3

1

3 5QNA(4UA)

4

1

4 5PET(3UA)

3

1

5 5PET(3UM)

3

1

6 5PET(3UU)

3

1

7 5PET(3URa)

3

1

8 5SUG(4UA)

4

1

TABLE 2 [CH₃—(OCH₂CH₂)_(n)—X¹]_(m)L—(X²—X³—R²)_(q) (I) Example No.Abbrev. Structure of Compound (I) 3 5QNA(3UA)

One of R^(X1), R^(X2), R^(X3) and R^(X4) is a hydrogen atom and theother three are CH₃—(OCH₂CH₂)_(n)—NH—C(═O)—. 8 5SUG(3UA)

One of R^(Y1), R^(Y2), R^(Y3) and R^(Y4) is a hydrogen atom and theother three are CH₃—(OCH₂OCH₂)_(n)—CH₂—NH—C(═O)—.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the blood half-life prolonging effect of chemicallymodified recombinant human interferon-β when intravenously injected intomice.

-   -▪-: change in the concentration of unmodified rhIFN-β in blood-   -▴-: change in the concentration of 5TRC(3UA)-rhIFN-β in blood-   -●-: change in the concentration of PEG₂Lys-rhIFN-β in blood

FIG. 2 shows the blood half-life prolonging effect of chemicallymodified recombinant human granulocyte-colony stimulating factors whenintravenously injected into rats.

-   -▪-: change in the concentration of unmodified rhG-CSF derivative in    blood-   -□-: change in the concentration of unmodified rhG-CSF in blood-   -▴-: change in the concentration of 5SKA(3UA)-rhG-CSF derivative in    blood-   -Δ-: change in the concentration of 5SKA(3UA)-rhG-CSF in blood-   -●-: change in the concentration of PEG₂Lys-rhG-CSF derivative in    blood-   -◯-: change in the concentration of PEG₂Lys-rhG-CSF in blood

BEST MODES FOR CARRYING OUT THE INVENTION

The present invention is specifically described by the followingexamples, which are not to be construed as limiting the scope of theinvention. The abbreviations in the examples mean the following unlessotherwise specified. The abbreviations for amino acids and theirprotective groups used herein follow the recommendations by IUPAC-IUBCommission on Biochemical Nomenclature [Eur. J. Biochem., Vol. 138, p. 9(1984)].

-   HPLC: high performance liquid chromatography-   RI: refractive index-   NMR: nuclear magnetic resonance-   ELISA: enzyme-linked immunosorbent assay-   SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis-   PEG: poly(ethylene glycol)-   mPEG: monomethoxy poly(ethylene glycol)-   IFN: interferon-   hIFN: human interferon-   rhIFN: recombinant human interferon-   G-CSF: granulocyte-colony stimulating factor-   rhG-CSF: recombinant human granulocyte-colony stimulating factor-   SOD: superoxide dismutase-   bSOD: bovine superoxide dismutase-   hSOD: human superoxide dismutase-   DSC: N,N′-disuccinimidyl carbonate-   TEA: triethylamine-   DMF: N,N-dimethylformamide-   DMSO: dimethyl sulfoxide-   NHS: N-hydroxysuccinimide-   Ts: p-toluenesulfonyl-   TsCl: p-toluenesulfonyl chloride-   DMAP: dimethylaminopyridine-   PyBOP: benzotriazol-1-yloxy-tripyrrolidinophosphonium    hexafluorophosphate-   HOBt: N-hydroxybenzotriazole-   DCC: N,N′-dicyclohexylcarbodiimide-   LAH: lithium aluminium hydride-   NMM: N-methylmorpholine-   TFA: trifluoroacetic acid-   CDI: N,N′-carbonyldiimidazole

EXAMPLE 1 Synthesis of 5 kDa Three-Chain Branched PolyethyleneGlycol-Tricine Derivative

Abbreviation: 5TRC(3UA)

In 0.5 ml of DMF were dissolved 0.5 mg (2.8 μmol) of tricine(N-[Tris(hydroxymethyl)methyl]glycine, Nacalai Tesque, Inc.) and 50 mg(10.0 μmol) of PEG-NCO (Shearwater Polymers, Inc., average molecularweight: 5,000, structure: CH₃(OCH₂CH₂)_(n)—N—C═O) in a stream of argon.To the solution were added 1.4 μl (10.0 μmol) of TEA and then ca. 1 mgof copper chloride, followed by stirring at room temperature for 5hours. To the mixture were further added 10 mg of PEG-NCO and 1 μl ofTEA, followed by stirring for 2 hours. Then, 15 mg of PEG-NCO was addedand the mixture was stirred a whole day and night at room temperature.

After addition of 50 ml of 0.1 mol/l hydrochloric acid, the mixture wasextracted with 50 ml of chloroform. The chloroform layer was dried overanhydrous sodium sulfate and the solvent was removed under reducedpressure. The residue was dissolved in a small amount of methylenechloride and the solution was added dropwise to diethyl ether. Theformed white precipitate was recovered by filtration to obtain 15 mg ofa crude product containing the desired compound (yield: 20%). Thisproduct was purified by DEAE Sepharose F.F. column (Amersham-PharmaciaBiotech). Elution was carried out with a 1 mol/l aqueous solution ofsodium chloride, and the eluate was extracted with chloroform underacidic conditions, followed by drying over anhydrous sodium sulfate.Thereafter, the solvent was removed under reduced pressure to obtain 6.0mg of the desired compound (yield: 8.0%).

<Gel Filtration HPLC Analysis>

The product was analyzed using TSKgelG2000SW_(XL) column (7.8×300 mm,Tosoh Corporation) under the following conditions.

-   Mobile phase: 150 mmol/l sodium chloride, 20 mmol/l sodium acetate    buffer (pH 4.5)-   Flow rate: 0.7 ml/minute-   Detection: R¹-   Retention time: 11.5 minutes

<¹H-NMR analysis (300 MHz, in CDCl₃)>

δ (ppm): 3.38(s, 9H), 3.64(s, 12 nH), 4.10(s, 6H), 5.43(br, 3H)

EXAMPLE 2

Synthesis of 5 kDa Three-Chain Branched Polyethylene Glycol-ShikimicAcid Derivative

Abbreviation: 5SKA(3UA)

In 250 μl of DMF was dissolved 3.2 mg of shikimic acid, and 15 μl oftriethylamine and a catalytic amount of copper chloride were addedthereto. To the mixture was added 300 mg of PEG-NCO (ShearwaterPolymers, Inc., average molecular weight: 5,000, structure:CH₃(OCH₂CH₂)_(n)—N—C═O), followed by stirring at room temperature forone hour. The reaction mixture was added dropwise to diethyl ether, andthe formed precipitate was recovered by filtration and dried underreduced pressure to obtain 270 mg (89%) of a crude desired product.

The product was purified using DEAE Sepharose F.F. column(Amersham-Pharmacia Biotech) in a manner similar to that in Example 1.The desired fraction was extracted with chloroform and the solvent wasremoved under reduced pressure to obtain 18 mg of the desired compound(yield: 6%).

<Gel Filtration HPLC Analysis>

Measurement was carried out using TSKgelG2000SW_(XL) column underconditions similar to those in Example 1. Retention time: 11.7 minutes

<¹H-NMR analysis (300 MHz, in CDCl₃)>

δ(ppm): 3.38(s, 9H), 3.64(s, 12 nH), 5.1-6.6(m, 4H)

EXAMPLE 3

Synthesis of 5 kDa Three- and Four-Chain Branched PolyethyleneGlycol-Quinic Acid Derivatives

Abbreviation: 5QNA(3UA), 5QNA(4UA)

In 250 μl of DMF was dissolved 3 mg of quinic acid ((1R, 3R, 4R,5R)-(−)-quinic acid), and 17 μl of triethylamine and a catalytic amountof copper chloride were added thereto. To the mixture was added 344 mgof PEG-NCO (Shearwater Polymers, Inc.), followed by stirring at roomtemperature for one hour. The reaction mixture was added dropwise todiethyl ether, and the formed precipitate was recovered by filtrationand dried under reduced pressure to obtain 306 mg (88%) of a crudedesired product. The product was purified using DEAE Sepharose F.F.column (Amersham-Pharmacia Biotech) in a manner similar to that inExample 1. The desired fraction was extracted with chloroform, and thesolvent was removed under reduced pressure to obtain the followingcompounds. TABLE 3 Retention Compound Number of Amount time in gelabbrev. PEG bound of product Yield filtration HPLC* 5QNA (3UA) 3 24 mg10.2% 11.7 minutes 5QNA (4UA) 4 17 mg 5.4% 11.1 minutes*Measurement was carried out using TSKgelG2000SW_(XL) column underconditions similar to those in Example 1.

<¹H-NMR analysis (300 MHz, in CDCl₃)>

Compound 5QNA(3UA): δ (ppm): 3.38(s, 9H), 3.64(s, 12 nH), 4.8-5.7(m, 3H)

Compound 5QNA(4UA): δ (ppm): 3.38(s, 12H), 3.64 (s, 16 nH) 4.8-5.7(m,3H)

EXAMPLE 4

Synthesis of 5 kDa Three-Chain Branched PolyethyleneGlycol-Pentaerythritol Derivative

Abbreviation: 5PET(3UA)

In 5 ml of DMF were dissolved 136 mg of pentaerythritol and 122 mg ofDMAP in a stream of argon, and 778 mg of CDI was added thereto. Themixture was stirred a whole day and night at 0° C. to room temperature.In 10 ml of DMF was dissolved 5.0 g of mPEG-NH₂ (NOF Corporation,average molecular weight: 5,000, structure: CH₃(OCH₂CH₂)_(n)—CH₂—NH₂),and 1.25 ml of the above reaction mixture was added thereto, followed bystirring at room temperature for 2 hours. A solution of 2.6 g ofγ-aminobutyric acid in 100 ml of 0.1 mol/l borate buffer (pH 10) wasice-cooled, and the reaction mixture was poured into this solution.After stirring at 0° C. for 2 hours and at room temperature for 4 hours,the mixture was made acidic with hydrochloric acid and then extractedwith chloroform. The solvent was removed under reduced pressure toobtain 4.2 g of a residue (84.6%). The residue (3.8 g) was purifiedusing DEAE Sepharose F.F. column (1000 ml, Amersham-Pharmacia Biotech)in a manner similar to that in Example 1 to obtain 254 mg of the desiredcompound (yield: 6.7%).

<Gel Filtration HPLC Analysis>

Measurement was carried out using TSKgelG2000SW_(XL) column underconditions similar to those in Example 1. Retention time: 11.4 minutes

<¹H-NMR analysis (300 MHz, in CDCl₃)>

δ (ppm): 5.44(brt, J=5.0 Hz, 3H), 5.25(br, 1H), 4.09(brs, 8H), 3.65(s,12 nH), 3.29(s, 9H), 3.26(m, 8H), 2.37(t, J=6.8 Hz, 2H), 1.80(brm, 2H),1.77(m, 6H)

EXAMPLE 5

Synthesis of 5 kDa Three-Chain Branched PolyethyleneGlycol-Pentaerythritol Derivative

Abbreviation: 5PET(3UM)

In 5 ml of DMF were dissolved 136 mg of pentaerythritol and 122 mg ofDMAP, and 778 mg of CDI was added thereto. The mixture was stirred awhole day and night at 0° C. to room temperature in a stream of argon.In 2 ml of DMF was dissolved 1.0 g of mPEG-NH₂ (NOF Corporation, averagemolecular weight: 5,000), and 0.25 ml of the above reaction mixture wasadded thereto, followed by stirring at room temperature for 2 hours.Then, 187 μl of propylenediamine was added thereto, and the mixture wasstirred at room temperature for 2 hours, followed by addition of diethylether. The formed white precipitate was recovered and dried underreduced pressure to obtain 975 mg of a residue (yield: 97.5%). Theresidue was purified using SP Sepharose F.F. column (100 ml,Amersham-Pharmacia Biotech), and the fraction eluted with 0.2 to 0.4mmol/l NaCl was extracted with chloroform to obtain 110 mg of a whitepowder (yield: 11.3%).

Subsequently, 100 mg of the white powder was dissolved in 0.5 ml of asaturated aqueous solution of sodium hydrogencarbonate, and 2.3 mg ofethoxycarbonyl maleimide was added thereto at 0° C., followed bystirring at 0° C. for 10 minutes. After addition of 1.5 ml of water, themixture was stirred at room temperature for 15 minutes and thenextracted with chloroform. The chloroform layer was concentrated underreduced pressure and added dropwise to diethyl ether. The formed whiteprecipitate was dried under reduced pressure to obtain 35 mg of thedesired compound (yield: 35%).

<Gel Filtration HPLC Analysis>

Measurement was carried out using TSKgelG2000SW_(XL) column underconditions similar to those in Example 1. Retention time: 11.3 minutes

<¹H-NMR analysis (300 MHz, in CDCl₃)>

δ (ppm): 6.73(s, 2H), 5.33(br, 3H), 4.08(brs, 8H), 3.64(s, 12 nH),3.36(s, 9H), 3.25(m, 6H), 3.11(m, 2H), 1.77(m, 8H)

EXAMPLE 6

Synthesis of Three-Chain Branched Polyethylene Glycol-PentaerythritolDerivative

Abbreviation: 5PET(3UU)

In 5 ml of DMF were dissolved 136 mg of pentaerythritol and 122 mg ofDMAP, and 681 mg of CDI was added thereto. The mixture was stirred awhole day and night at 0° C. to room temperature in a stream of argon.In 2 ml of DMF was dissolved 1.0 g of mPEG-NH₂ (NOF Corporation, averagemolecular weight: 5,000), and 286 μl of the above reaction mixture wasadded thereto, followed by stirring at room temperature for 2 hours. Theresulting reaction mixture was added dropwise to diethyl ether, and theformed white precipitate was recovered and dried under reduced pressureto obtain 1 g of a residue (yield: 100%).

The residue was purified using TSKgelODS-120T column (30 mm×250 mm,Tosoh Corporation). As an eluent, 0 to 90% aqueous acetonitrile solutioncontaining 0.1% TFA was used. The fraction containing three-chain PEGwas concentrated under reduced pressure and extracted with chloroform,and the solvent was removed under reduced pressure to obtain 165 mg of aresidue (yield: 16.5%).

The obtained white powder (80 mg) was dissolved in 1 ml of methylenechloride, and 4.1 mg of DSC and 2.1 mg of DMAP were added thereto,followed by stirring at room temperature for 6 hours in a stream ofargon. The reaction mixture was added dropwise to diethyl ether, and theformed white precipitate was dried under reduced pressure to obtain 63mg of the desired compound (yield: 78.8%).

<Gel Filtration HPLC Analysis>

Measurement was carried out using TSKgelG2000SW_(XL) column underconditions similar to those in Example 1. Retention time: 10.7 minutes

<¹H-NMR analysis (300 MHz, in CDCl₃)>

δ (ppm): 5.49(br, 3H), 4.11(brs, 8H), 3.64(s, 12 nH), 3.38(s, 9H),3.25(m, 6H), 2.87(s, 4H), 1.78(m, 8H)

EXAMPLE 7

Synthesis of Three-Chain Branched Polyethylene Glycol-PentaerythritolDerivative

Abbreviation: 5PET(3URa).

In 5 ml of DMF were dissolved 136 mg of pentaerythritol and 122 mg ofDMAP, and 681 mg of CDI was added thereto. The mixture was stirred awhole day and night at 0° C. to room temperature in a stream of argon.In 2 ml of DMF was dissolved 1.0 g of mPEG-NH₂ (NOF Corporation, averagemolecular weight: 5,000), and 286 μl of the above reaction mixture wasadded thereto, followed by stirring at room temperature for 2 hours. Theresulting reaction mixture was added dropwise to diethyl ether, and theformed white precipitate was recovered and dried under reduced pressureto obtain 950 mg of a residue (yield: 95%). The residue was purifiedusing TSKgelODS-120T column (30 mm×250 mm, Tosoh Corporation). As aneluent, 0 to 90% aqueous acetonitrile solution containing 0.1% TFA wasused. The fraction containing three-chain PEG was concentrated underreduced pressure and extracted with chloroform, and the solvent wasremoved under reduced pressure to obtain 300 mg of a residue (yield:31.6%).

The obtained residue (white powder, 300 mg) was dissolved in 1 ml ofmethylene chloride, and 15.4 mg of DSC and 7.3 mg of DMAP were addedthereto, followed by stirring at room temperature for 6 hours in astream of argon. The reaction mixture was added dropwise to diethylether, and the formed white precipitate was dried under reducedpressure. The resulting dried product was dissolved in 1 ml of methylenechloride, and 3.5 μl of 4-aminobutyraldehyde diethylacetal was addedthereto, followed by stirring at room temperature for 2 hours. Thereaction mixture was added dropwise to diethyl ether, and the formedwhite precipitate was dried under reduced pressure to obtain 250 mg of aresidue (yield: 83.3%).

The obtained residue (100 mg) was dissolved in methylene chloridecontaining 10% TFA, and the solution was allowed to stand at 0° C. forone hour. Then, the solution was added dropwise to diethyl ether, andthe formed white precipitate was dried under reduced pressure to obtain40 mg of the desired compound (yield: 40.0%).

<Gel Filtration HPLC Analysis>

Measurement was carried out using TSKgelG2000SW_(XL) column underconditions similar to those in Example 1. Retention time: 10.6 minutes

EXAMPLE 8

Synthesis of Three- and Four-Chain Branched PolyethyleneGlycol-Carbohydrate Derivatives

Abbreviation: 5SUG(3UA), 5SUG(4UA)

In 80 ml of DMF was dissolved 5.18 g of α-D-glucose pentaacetate, and2.37 g of hydrazine acetate was added thereto, followed by stirring atroom temperature for 1.5 hours. The reaction mixture was extracted withethyl acetate, and the ethyl acetate layer was washed with water and asaturated aqueous solution of sodium chloride, and then dried overanhydrous sodium sulfate. The solution was concentrated under reducedpressure to obtain 4.0 g of α-D-glucopyranose-2,3,4,6-tetraacetate(yield: 87%).

<¹H-NMR analysis (300 MHz, in CDCl₃)>

δ (ppm): 2.02(s, 3H), 2.03(s, 3H), 2.08(s, 3H), 2.10(s, 3H), 4.14(m,1H), 4.27(m, 2H), 4.91(m, 1H), 5.09(t, J=9.7 Hz, 1H), 5.47(d, J=3.7 Hz,1H), 5.55(t, J=9.8 Hz, 1H)

The above compound (850 mg) was dissolved in 15 ml of methylenechloride, and 4.8 ml of trichloroacetonitrile and 365 ml of DBU(1,8-diazabicyclo[5.4.0]undec-7-ene) were added thereto at 0° C.,followed by stirring at 0° C. for one hour and at room temperature for15 minutes. The resulting solution was concentrated under reducedpressure and then purified using a silica gel column to obtain 635 mg ofα-D-glucopyranose-2,3,4,6-tetraacetate-1-(2,2,2-trichloroethanimidate)(yield: 53%).

<¹H-NMR analysis (CDCl₃, 300 MHz)>

δ (ppm): 2.02(s, 3H), 2.04(s, 3H), 2.06(s, 3H), 2.08(s, 3H), 4.13(m,1H), 4.21(m, 1H), 4.28(m, 1H), 5.13(m, 1H), 5.19(t, J=9.8 Hz, 1H),5.57(t, J=9.9 Hz, 1H), 6.56(d, J=3.7 Hz, 1H), 8.71(s, 1H)

The above compound (693 mg) and 109 μl of methyl glycolate weredissolved in dehydrated methylene chloride, and 1.62 g of molecularsieves 4A was added thereto, followed by stirring at room temperaturefor 4 hours in a stream of argon. The reaction mixture was cooled to 0to 5° C., and 163 μl of a mixed solution of trimethylsilyltrifluoromethanesulfonate and dehydrated methylene chloride (2:1) wasadded thereto, followed by stirring at 0 to 5° C. for 19 hours. Afteraddition of 77 μl of triethylamine, the mixture was filtered throughCelite. The resulting solution was concentrated under reduced pressureand then purified using a silica gel column to obtain 162 mg of[(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)oxy] acetic acid methylester (yield: 27%).

<¹H-NMR analysis (CDCl₃, 300 MHz)>

δ (ppm): 2.01(s, 3H), 2.03(s, 3H), 2.09(s, 3H), 2.10(s, 3H), 3.70(m,1H), 3.75(s, 3H), 4.14(m, 1H), 4.26(m, 1H), 4.29(s, 2H), 4.67(d, J=7.8Hz, 1H), 5.05(m, 1H), 5.09(t, J=10.8 Hz, 1H), 5.25(t, J=9.5 Hz, 1H) Theabove compound (162 mg) was dissolved in 1 ml of methanol, and Amberlystwas added thereto. Then, 9.4 μl of a 28% solution of sodium methoxide inmethanol was added, and the mixture was stirred at room temperature for3 hours. After filtration through Celite, the filtrate was concentratedunder reduced pressure to obtain 80 mg of [(β-D-glucopyranosyl)oxy]acetic acid methyl ester (yield: 82%).

<¹H-NMR analysis (D₂O, 300 MHz)>

δ (ppm): 3.39(s, 2H), 3.40(m, 2H), 3.69(m, 1H), 3.75(s, 3H), 3.86(m,1H), 4.06(m, 1H), 4.26(m, 1H), 4.44(m, 1H)

<Mass spectrum (FAB-MS)>

Found: [M+H]=253

Calcd.: C₉H₁₆O₈=252

The above compound (2 mg) was dissolved in 100 μl of DMF, and 7 μl oftriethylamine and a catalytic amount of CuCl were added thereto. To themixture was added 160 mg of mPEG-NCO, and the mixture was stirred atroom temperature for 2 hours. Then, 80 mg of mPEG-NCO was added,followed by further stirring for 3 hours. The resulting solution wasadded dropwise to diethyl ether, and the formed white precipitate wasrecovered by filtration and dried under reduced pressure. The obtainedwhite solid (200 mg) was dissolved in 2 ml of 1 mol/l aqueous solutionof potassium carbonate, followed by stirring at room temperature for 4hours. To the solution were added chloroform and 0.1 mol/l hydrochloricacid, and the mixture was extracted with chloroform. After the extractwas dried over anhydrous sodium sulfate, the solvent was removed underreduced pressure, and the residue was dried under reduced pressure toobtain 195 mg of a white solid. This product was purified using DEAESepharose F.F. column (20 ml, Amersham-Pharmacia Biotech) to obtain thecompounds shown below. TABLE 4 Compound Number of Amount of Retentiontime in gel abbrev. PEG bound product Yield filtration HPLC* 5SUG (3UA)3 6 mg 5.0% 10.8 minutes 5SUG (4UA) 4 12 mg 7.6% 10.4 minutes*Measurement was carried out using TSKgelG2000SW_(XL) column underconditions similar to those in Example 1.

<¹H-NMR analysis (300 MHz, in CDCl₃)>

Compound 5SUG(3UA): δ (ppm): 3.38 (s, 9H), 3.64(t, 12 nH) 4.1-5.6(m, 7H)

Compound 5SUG(4UA): δ (ppm): 3.38(s, 12H), 3.64(t, 16 nH), 4.1-5.6(m,7H)

EXAMPLE 9

Preparation of Recombinant Human Interferon-β Modified with 5 KDaThree-Chain Branched Polyethylene Glycol

Abbreviation: 5TRC(3UA)-rhIFN-β

To 5 mg (0.33 μmol) of the compound of Example 1 (5TRC(3UA)) were added50 μl (0.66 μmol) of 1.5 mg/ml solution of NHS in methylene chloride and100 μl (0.66μmol) of 1.4 mg/ml solution of DCC in methylene chloride,followed by stirring in a stream of argon under ice-cooling for 30minutes and at room temperature for 2 hours. After addition of diethylether, the formed precipitate was dried under reduced pressure to obtain3.5 mg (yield: 70%) of NHS ester.

To 150 μl of a 0.9 mg/ml solution of rhIFN-β obtained in ReferenceExample 4 in 20 mmol/l phosphate buffer containing ethylene glycol andsodium chloride was added 33.4 mg (34 mol per mol of protein) of themodifying reagent activated above (NHS ester), and the mixture wassubjected to reaction by standing a whole day and night at 4° C. Thereaction mixture was applied to a gel filtration column Sephadex G-25(Amersham-Pharmacia Biotech) and subjected to buffer exchange with 20mmol/l phosphate buffer (pH 6.0) containing ethylene glycol, followed bypurification using CM Sepharose F.F. column (0.5 ml, Amersham-PharmaciaBiotech). After the reaction mixture was charged, the column was washedwith 5 ml of the same buffer, and elution was carried out with thebuffer containing sodium chloride. The fraction containing the desiredsubstance was recovered to obtain 0.40 ml of the desired substance(0.091 mg/ml) (yield: 27.0%).

<Electrophoresis>

SDS-PAGE was carried out in the presence of 2-mercaptoethanol under thefollowing conditions to confirm the bands of 1 to 3 molecules-boundsubstances.

Gel: PAGEL SPG 520L (Atto Corporation)

Staining: FAST STAIN™

Molecular weight marker: Low Molecular Weight Standard

(Bio-Rad)

<Gel Filtration HPLC Analysis>

Analysis was carried out using two TSKgelG4000SW_(XL) columns under thefollowing conditions.

Mobile phase: 150 mmol/l sodium chloride, 20 mmol/l sodium acetatebuffer (pH 4.5)

Flow rate: 0.5 ml/minute

Detection: UV 280 nm

Separation column: TSKgelG4000SW_(XL) (7.8×300 mm×2, Tosoh Corporation)

Retention time: 42.0 minutes (1 molecule-bound substance) 44.1 minutes(2 molecules-bound substance)

EXAMPLE 10

Preparation of Recombinant Human Interferon-β Modified with 5 kDaThree-Chain Branched Polyethylene Glycol

Abbreviation: 5SKA(3UA)-rhIFN-β

In 100 μl of methylene chloride was dissolved 16 mg (1.1 μmol) of thecompound of Example 2 (5SKA(3UA)), and 272 μg of DCC and 152 μg of NHSwere added thereto, followed by stirring under ice-cooling for one hourand at room temperature for one hour. The mixture was added dropwise todiethyl ether, and the formed white precipitate was dried under reducedpressure to obtain 14.5 mg of NHS ester of the compound of Example 2(yield: 91%).

To 100 μl of a 1.2 mg/ml solution of rhIFN-β obtained in ReferenceExample 4 in 20 mmol/l phosphate buffer containing ethylene glycol andsodium chloride was added 8.6 mg (100 mol per mol of protein) of the NHSester obtained above, and the mixture was subjected to reaction bystanding a whole day and night at 4° C. The reaction mixture was appliedto a gel filtration column Sephadex G-25 (Amersham-Pharmacia Biotech)and subjected to buffer exchange with 20 mmol/l phosphate buffer (pH6.0) containing ethylene glycol, followed by purification using CMSepharose F.F. column (0.6 ml, Amersham-Pharmacia Biotech). After thereaction mixture was charged, the column was washed with 3 ml of thesame buffer, and elution was carried out with the buffer containingsodium chloride. The fraction containing the desired substance wasrecovered to obtain 80 μl of the desired substance (47 μg/ml) (yield:3.3%).

<Electrophoresis>

SDS-PAGE was carried out in the presence of 2-mercaptoethanol in amanner similar to Example 9 to confirm the band of 1 molecule-boundsubstance.

<Gel Filtration HPLC Analysis>

Analysis was carried out using two TSKgelG4000SW_(XL) columns in amanner similar to Example 9.

Retention time: 41.7 minutes (1 molecule-bound substance)

EXAMPLE 11

Preparation of Recombinant Human Interferon-β Modified with 5 kDaThree-Chain Branched Polyethylene Glycol

Abbreviation: 5PET(3UU)-rhIFN-β

To 0.5 ml of a 1.2 mg/ml solution of rhIFN-β obtained in ReferenceExample 4 in 20 mmol/l phosphate buffer (pH 7.8) containing ethyleneglycol and sodium chloride was added 4.5 mg (10 mol per mol of protein)of 5PET(3UU) obtained in Example 6, and the mixture was subjected toreaction a whole day and night at 4° C. The reaction mixture (0.5 ml)was applied to Sephadex G-25 column (Amersham-Pharmacia Biotech) andsubjected to buffer exchange with 20 mmol/l phosphate buffer (pH 6)containing ethylene glycol. The mixture was passed through CM-SepharoseF.F. column (0.8 ml, Amersham-Pharmacia Biotech), followed by washingwith 4.0 ml of 20 mmol/l phosphate buffer (pH 6) containing ethyleneglycol. Elution was carried out with the same buffer containing 0.1 to0.5 mol/l sodium chloride, and the desired fractions were combined andthen concentrated to obtain 0.36 ml of a solution containing the desiredsubstance (0.67 mg/ml) (yield: 40%).

<Electrophoresis>

SDS-PAGE was carried out in the presence of 2-mercaptoethanol in amanner similar to Example 9 to confirm the bands of 1 to 3molecules-bound substances.

<Gel Filtration HPLC Analysis>

Analysis was carried out using two TSKgelG4000SW_(XL) columns in amanner similar to Example 9. Retention time: 41.1 minutes (1molecule-bound substance) 38.2 minutes (2 molecules-bound substance)

EXAMPLE 12

Preparation of Recombinant Human Interferon-β Modified with 5 kDaThree-Chain Branched Polyethylene Glycol

Abbreviation: 5PET(3UA)-rhIFN-β

In 2.0 ml of methylene chloride was dissolved 254 mg (0.02 mmol) of thecompound of Example 4 (5PET(3UA)), and 5.9 mg (0.05 mmol) of NHS and10.5 mg (0.05 mmol) of DCC were added thereto, followed by stirring in astream of argon at 0° C. for one hour and at room temperature for 2hours. The reaction mixture was added dropwise to diethyl ether, and theformed white precipitate was dried under reduced pressure to obtain132.8 mg of NHS ester of the compound of Example 4 (yield: 52.3%).

To 1.0 ml of a 1.16 mg/ml solution of rhIFN-β obtained in ReferenceExample 4 in 20 mmol/l phosphate buffer (pH 7.8) containing ethyleneglycol and sodium chloride was added 13 mg (15 mol per mol of protein)of the above NHS ester of 5PET(3UA), and the mixture was subjected toreaction a whole day and night at 4° C. The reaction mixture was appliedto Sephadex G-25 column (Amersham-Pharmacia Biotech) and subjected tobuffer exchange with 20 mmol/l phosphate buffer (pH 6) containingethylene glycol. The mixture was passed through CM-Sepharose F.F. column(1.4 ml, Amersham-Pharmacia Biotech), followed by washing with 7.0 ml of20 mmol/l phosphate buffer (pH 6) containing ethylene glycol. Elutionwas carried out with the same buffer containing 0.1 to 0.5 mol/l sodiumchloride, and the desired fractions were combined and then concentratedto obtain 1.0 ml of a solution containing the desired substance (0.14mg/ml) (yield: 12%).

<Electrophoresis>

SDS-PAGE was carried out in the presence of 2-mercaptoethanol in amanner similar to Example 9 to confirm the bands of 1 to 3molecules-bound substances.

<Gel Filtration HPLC Analysis>

Analysis was carried out using two TSKgelG4000SW_(XL) columns in amanner similar to Example 9.

Retention time: 43.8 minutes (1 molecule-bound substance) 41.2 minutes(2 molecules-bound substance)

EXAMPLE 13

Preparation of Recombinant Human Interferon-β Modified with 5 kDaThree-Chain Branched Polyethylene Glycol

Abbreviation: 5PET(3UA)-¹⁷Ser rhIFN-β

To 0.05 ml of a 2.1 mg/ml solution of ¹⁷Ser rhIFN-β (Chiron) in 20mmol/l phosphate buffer (pH 7.5) containing ethylene glycol and sodiumchloride was added 1.6 mg (20 mol per mol of protein) of NHS ester of5PET(3UA) obtained in a manner similar to Example 12, and the mixturewas subjected to reaction a whole day and night at 4° C. The reactionmixture was applied to Sephadex G-25 column (Amersham-Pharmacia Biotech)and subjected to buffer exchange with 20 mmol/l phosphate buffer (pH 6)containing ethylene glycol. The fraction obtained by gel filtration waspassed through CM-Sepharose F.F. column (0.5 ml, Amersham-PharmaciaBiotech), followed by washing with 8 ml of 20 mmol/l phosphate buffer(pH 6) containing ethylene glycol. Elution was carried out with the samebuffer containing 0.2 to 1.0 mol/l sodium chloride, and the desiredfractions were combined and then concentrated to obtain 0.30 ml of asolution containing the desired substance (27.8 μg/ml) (yield: 7.9%).

<Electrophoresis>

SDS-PAGE was carried out in the presence of 2-mercaptoethanol in amanner similar to Example 9 to confirm the bands of 1 to 3molecules-bound substances.

EXAMPLE 14

Preparation of Recombinant Human Interferon-α Modified with 5 kDaThree-Chain Branched Polyethylene Glycol

Abbreviation: 5PET(3UA)-rhIFN-α

To 0.1 ml of a 1.0 mg/ml solution of rhIFN-α (IBL Co., Ltd.) in isotonicphosphate buffer (pH 7.5) was added 1.6 mg (20 mol per mol of protein)of NHS ester of 5PET(3UA) obtained in a manner similar to Example 12,and the mixture was subjected to reaction a whole day and night at 4° C.The reaction mixture was applied to Sephadex G-25 column(Amersham-Pharmacia Biotech) and subjected to buffer exchange with 20mmol/l sodium acetate buffer (pH 4.5). The mixture was passed throughSP-Sepharose F.F. column (0.7 ml, Amersham-Pharmacia Biotech), followedby washing with 20 mmol/l sodium acetate buffer (pH 4.5). Elution wascarried out with the same buffer containing 0.1 to 0.5 mol/l sodiumchloride, and the desired fractions were combined and then concentratedto obtain 65 μl of a solution containing the desired substance (0.53mg/ml) (yield: 34%).

<Electrophoresis>

SDS-PAGE was carried out in the presence of 2-mercaptoethanol in amanner similar to Example 9 to confirm the bands of 1 to 3molecules-bound substances.

<Gel Filtration HPLC Analysis>

Analysis was carried out using two TSKgelG4000SW_(XL) columns in amanner similar to Example 9.

Retention time: 42.6 minutes (1 molecule-bound substance) 40.3 minutes(2 molecules-bound substance)

EXAMPLE 15

Preparation of Recombinant Human Granulocyte-Colony Stimulating FactorDerivative Modified with 5 KDa Three-Chain Branched Polyethylene Glycol

Abbreviation: 5SKA(3UA)-rhG-CSF derivative

In 100 μl of methylene chloride was dissolved 16 mg (1.1 μmol) of thecompound of Example 2 (5SKA(3UA)), and 272 μg of DCC and 152 μg of NHSwere added thereto, followed by stirring under ice-cooling for one hourand at room temperature for one hour. The reaction mixture was addeddropwise to diethyl ether, and the formed white precipitate was driedunder reduced pressure to obtain 14.5 mg of NHS ester of the compound ofExample 2 (yield: 91%).

To 50 μl of a 3.7 mg/ml solution of the rhG-CSF derivative obtained inReference Example 5 in 50 mmol/l phosphate buffer (pH 7.5) was added 3.6mg (25 mol per mol of protein) of the compound activated above (NHSester), and the mixture was subjected to reaction a whole day and nightat 4° C. The reaction mixture was applied to Sephadex G-25 column(Amersham-Pharmacia Biotech) and subjected to buffer exchange with 20mmol/l acetate buffer (pH 4.5), followed by purification using SPSepharose F.F. column (0.7 ml, Amersham-Pharmacia Biotech). The desiredfraction was concentrated to obtain 165 μl of a solution containing thedesired substance (0.4 mg/ml) (yield: 36%).

<Electrophoresis>

SDS-PAGE was carried out in the absence of 2-mercaptoethanol in a mannersimilar to Example 9 to confirm the bands of 1 to 3 molecules-boundsubstances.

<Gel Filtration HPLC Analysis>

Analysis was carried out using two TSKgelG4000SW_(XL) columns in amanner similar to Example 9.

Retention time: 42.3 minutes (1 molecule-bound substance) 40.2 minutes(2 molecules-bound substance)

EXAMPLE 16

Preparation of a Solution Containing Recombinant HumanGranulocyte-Colony Stimulating Factor Modified with 5 KDa Four-ChainBranched Polyethylene Glycol

Abbreviation: 5QNA(4UA)-rhG-CSF derivative

In 500 μl of methylene chloride was dissolved 69 mg (3.5 mmol) of thecompound of Example 3 (5QNA(4UA)), and 1.8 mg of DSC and 0.56 mg of DMAPwere added thereto, followed by stirring at room temperature for 6hours. The reaction mixture was added dropwise to diethyl ether, and theformed white precipitate was dried under reduced pressure to obtain 44mg of NHS ester of the compound of Example 3 (yield: 63%).

To 50 μl of a 3.8 mg/ml solution of the rhG-CSF derivative obtained inReference Example 5 in 50 mmol/l phosphate buffer (pH 8) was added 5.1mg (25 mol per mol of protein) of the compound activated above (NHSester), and the mixture was subjected to reaction a whole day and nightat 4° C. Without further purification steps, the resulting product wasconfirmed by electrophoresis and gel filtration HPLC analysis.

<Electrophoresis>

SDS-PAGE was carried out in the absence of 2-mercaptoethanol in a mannersimilar to Example 9 to confirm the band of 1 molecule-bound substance.

<Gel Filtration HPLC Analysis>

Analysis was carried out using two TSKgelG4000SW_(XL) columns in amanner similar to Example 9.

Retention time: 40.8 minutes (1 molecule-bound substance)

EXAMPLE 17

Preparation of Recombinant Human Granulocyte-Colony Stimulating FactorModified with 5 KDa Three-Chain Branched Polyethylene Glycol

Abbreviation: 5SKA(3UA)-rhG-CSF

In 100 μl of methylene chloride was dissolved 16 mg (1.1 μmol) of thecompound of Example 2 (5SKA(3UA)), and 272 μg of DCC and 152 μg of NHSwere added thereto, followed by stirring under ice-cooling for one hourand at room temperature for one hour. The reaction mixture was addeddropwise to diethyl ether, and the formed white precipitate was driedunder reduced pressure to obtain 14.5 mg of NHS ester of the compound ofExample 2 (yield: 91%).

To 140 μl of a 4.4 mg/ml solution of the rhG-CSF obtained in ReferenceExample 6 in 50 mmol/l phosphate buffer (pH 7.5) was added 12.2 mg (25mol per mol of protein) of the compound activated above (NHS ester), andthe mixture was subjected to reaction a whole day and night at 4° C. Thereaction mixture was applied to Sephadex G-25 column (Amersham-PharmaciaBiotech) and subjected to buffer exchange with 20 mmol/l acetate buffer(pH 4.5), followed by purification using SP Sepharose F.F. column (1.8ml, Amersham-Pharmacia Biotech). The desired fraction was concentratedto obtain 110 μl of a solution containing the desired substance (1.1mg/ml) (yield: 19%).

<Electrophoresis>

SDS-PAGE was carried out in the absence of 2-mercaptoethanol in a mannersimilar to Example 9 to confirm the bands of 1 to 3 molecules-boundsubstances.

<Gel Filtration HPLC Analysis>

Analysis was carried out using two TSKgelG4000SW_(XL) columns in amanner similar to Example 9.

Retention time: 40 to 45 minutes (1 to 3 molecules-bound substances)

EXAMPLE 18 Preparation of Recombinant Human Granulocyte-ColonyStimulating Factor Derivative Modified with 5 KDa Three-Chain BranchedPolyethylene Glycol

Abbreviation: 5PET(3UU)-rhG-CSF derivative

To 0.5 ml of a 3.1 mg/ml solution of the rhG-CSF derivative obtained inReference Example 5 in 20 mmol/l phosphate buffer (pH 7.5) was added12.2 mg (10 mol per mol of protein) of 5PET(3UU) obtained in Example 6,and the mixture was subjected to reaction a whole day and night at 4° C.The reaction mixture was applied to Sephadex G-25 column(Amersham-Pharmacia Biotech) and subjected to buffer exchange with 20mmol/l sodium acetate buffer (pH 4.5). The mixture was passed throughSP-Sepharose F.F. column (1.5 ml, Amersham-Pharmacia Biotech), followedby washing with 7.5 ml of 20 mmol/l sodium acetate buffer (pH 4.5).Elution was carried out with the same buffer containing 0.2 to 0.5 mol/lsodium chloride, and the desired fractions were combined and thenconcentrated to obtain 0.75 ml of a solution containing the desiredsubstance (1.2 mg/ml) (yield: 58.6%).

<Electrophoresis>

SDS-PAGE was carried out in the absence of 2-mercaptoethanol in a mannersimilar to Example 9 to confirm the bands of 1 to 3 molecules-boundsubstances.

<Gel Filtration HPLC Analysis>

Analysis was carried out using two TSKgelG4000SW_(XL) columns in amanner similar to Example 9.

Retention time: 40.5 minutes (1 molecule-bound substance) 37.8 minutes(2 molecules-bound substance)

EXAMPLE 19

Preparation of Recombinant Human Granulocyte-Colony Stimulating FactorDerivative Modified with 5 KDa Three-Chain Branched Polyethylene Glycol

Abbreviation: 5PET(3UA)-rhG-CSF Derivative

To 0.05 ml of a 4.0 mg/ml solution of the rhG-CSF derivative obtained inReference Example 5 in 20 mmol/l phosphate buffer (pH 7.5) was added 1.6mg (10 mol per mol of protein) of NHS ester of 5PET(3UA) obtained in amanner similar to Example 12, and the mixture was subjected to reactiona whole day and night at 4° C. The reaction mixture was applied toSephadex G-25 column (Amersham-Pharmacia Biotech) and subjected tobuffer exchange with 20 mmol/l sodium acetate buffer (pH 4.5). Themixture was passed through SP-Sepharose F.F. column (0.7 ml,Amersham-Pharmacia Biotech), followed by washing with 20 mmol/l sodiumacetate buffer (pH 4.5). Elution was carried out with the same buffercontaining 0.2 to 0.5 mol/l sodium chloride, and the desired fractionswere combined and then concentrated to obtain 0.30 ml of a solutioncontaining the desired substance (0.34 mg/ml) (yield: 56.7%).

<Electrophoresis>

SDS-PAGE was carried out in the absence of 2-mercaptoethanol in a mannersimilar to Example 9 to confirm the bands of 1 to 3 molecules-boundsubstances.

<Gel Filtration HPLC Analysis>

Analysis was carried out using two TSKgelG4000SW_(XL) columns in amanner similar to Example 9.

Retention time: 42.3 minutes (1 molecule-bound substance) 39.5 minutes(2 molecules-bound substance)

EXAMPLE 20

Preparation of recombinant human granulocyte-colony stimulating factorderivative modified with 5 kDa three-chain branched polyethylene glycol

Abbreviation: 5SUG(3UA)-rhG-CSF Derivative

To 100 mg (6.7 μmol) of the compound obtained in Example 8 (5SUG(3UA))were added 2.3 mg of NHS and 4.1 mg of DCC, and the mixture wasdissolved in 1 ml of methylene chloride under ice-cooling, followed bystirring under ice-cooling for one hour and at room temperature for 1.5hours. The reaction mixture was added dropwise to diethyl ether and theformed white precipitate was dried under reduced pressure to obtain 76.6mg of NHS ester of the compound of Example 8 (yield: 76.6%).

To 0.1 ml of a 3.9 mg/ml solution of the rhG-CSF derivative obtained inReference Example 5 in 50 mmol/l phosphate buffer (pH 7.5) was added10.7 mg (35 mol per mol of protein) of the compound activated above (NHSester), and the mixture was subjected to reaction a whole day and nightat 4° C. The reaction mixture was applied to Sephadex G-25 column(Amersham-Pharmacia Biotech) and subjected to buffer exchange with 20mmol/l sodium acetate buffer (pH 4.5). The mixture was passed throughSP-Sepharose F.F. column (0.7 ml, Amersham-Pharmacia Biotech), followedby washing with 20 mmol/l sodium acetate buffer (pH 4.5). Elution wascarried out with the same buffer containing 0.2 to 0.5 mol/l sodiumchloride, and the desired fractions were combined and then concentratedto obtain 0.39 ml of a solution containing the desired substance (0.28mg/ml) (yield: 27.8%).

<Electrophoresis>

SDS-PAGE was carried out in the absence of 2-mercaptoethanol in a mannersimilar to Example 9 to confirm the bands of 1 to 3 molecules-boundsubstances.

<Gel Filtration HPLC Analysis>

Analysis was carried out using two TSKgelG4000SW_(XL) columns in amanner similar to Example 9.

Retention time: 43.0 minutes (1 molecule-bound substance) 40.4 minutes(2 molecules-bound substance)

EXAMPLE 21

Preparation of Human Cu, Zn-Superoxide Dismutase Modified with 5 kDaThree-Chain Branched Polyethylene Glycol

Abbreviation: 5PET(3UM)-hSOD

To 0.5 ml of a 1.34 mg/ml solution of Cu, Zn-hSOD (CELLULAR PRODUCTS,INC.) in 50 mmol/l phosphate buffer (pH 7.5) was added 3.1 mg (10 molper mol of protein) of 5PET(3UM) obtained in Example 5, and the mixturewas subjected to reaction a whole day and night at 4° C. The reactionmixture was applied to Sephadex G-25 column (Amersham-Pharmacia Biotech)and subjected to buffer exchange with 20 mmol/l sodium acetate buffer(pH 3.5). The mixture was passed through SP-Sepharose F.F. column (0.7ml, Amersham-Pharmacia Biotech), followed by washing with 20 mmol/lsodium acetate buffer (pH 3.5). Elution was carried out with the samebuffer containing 0.5 to 1.0 mol/l sodium chloride, and the desiredfractions were combined and then concentrated to obtain 0.62 ml of asolution containing the desired substance (0.33 mg/ml) (yield: 30.6%).

<Electrophoresis>

SDS-PAGE was carried out in the absence of 2-mercaptoethanol in a mannersimilar to Example 9 to confirm the band of 1 molecule-bound substance.

<Gel Filtration HPLC Analysis>

Analysis was carried out using two TSKgelG4000SW_(XL) columns in amanner similar to Example 9.

Retention time: 41.1 minutes (1 molecule-bound substance)

EXAMPLE 22

Preparation of Anti-GD3 Chimera Antibody Modified with 5 kDa Three-ChainBranched Polyethylene Glycol

Abbreviation: 5PET(3UA)-KM871

To 1.0 ml of a 1.1 mg/ml solution of anti-GD3 chimera antibody (KM-871)in 20 mmol/l phosphate buffer (pH 7.5) (prepared according to JapanesePublished Unexamined Patent Application No. 304989/93) was added 0.6 mg(5 mol per mol of protein) of NHS ester of 5PET(3UA) obtained in amanner similar to Example 12, and the mixture was subjected to reactiona whole day and night at 40C. The reaction mixture (1.0 ml) was appliedto Sephadex G-25 column (Amersham-Pharmacia Biotech) and subjected tobuffer exchange with 20 mmol/l acetate buffer (pH 4.5). The mixture waspassed through CM-Sepharose F.F. column (1.0 ml, Amersham-PharmaciaBiotech), followed by washing with 20 mmol/l sodium acetate buffer (pH4.5). Elution was carried out with the same buffer containing 0.25 to1.0 mol/l sodium chloride, and the desired fractions were combined andthen concentrated to obtain 430 μl of a solution containing the desiredsubstance (0.52 mg/ml) (yield: 20.4%).

<Electrophoresis>

SDS-PAGE was carried out in the absence of 2-mercaptoethanol in a mannersimilar to Example 9 to confirm the bands of 1 to 2 molecules-boundsubstances.

EXAMPLE 23

Preparation of Recombinant Human Granulocyte-Colony Stimulating FactorDerivative Modified with 5 KDa Three-Chain Branched Polyethylene Glycol

Abbreviation: 5PET(3URa)-rhG-CSF derivative

To 0.6 ml of a 2.35 mg/ml solution of the rhG-CSF derivative obtained inReference Example 5 in 50 mmol/l phosphate buffer (pH 7.5) were added56.3 mg (50 mol per mol of protein) of the compound of Example 7(5PET(3URa)) and 10 μl of a 120 mmol/l aqueous NaBH₃CN solution. Themixture was subjected to reaction a whole day and night at 4° C. andthen made acidic with hydrochloric acid to stop the reaction. Thereaction mixture was applied to Sephadex G-25 column (Amersham-PharmaciaBiotech) and subjected to buffer exchange with 20 mmol/l sodium acetatebuffer (pH 4.5). The mixture was passed through SP-Sepharose F.F. column(1.4 ml, Amersham-Pharmacia Biotech), followed by washing with 20 mmol/lsodium acetate buffer (pH 4.5). Elution was carried out with the samebuffer containing 0.1 to 0.2 mol/l sodium chloride, and the desiredfractions were combined and then concentrated to obtain 0.55 ml of asolution containing the desired substance (0.24 mg/ml) (yield: 8.5%).

<Electrophoresis>

SDS-PAGE was carried out in the absence of 2-mercaptoethanol in a mannersimilar to Example 9 to confirm the band of 1 molecule-bound substance.

<Gel Filtration HPLC Analysis>

Analysis was carried out using two TSKgelG4000SW_(XL) columns in amanner similar to Example 9.

Retention time: 41.2 minutes (1 molecule-bound substance)

REFERENCE EXAMPLE 1

Preparation of Recombinant Human Interferon-β Modified with 5 kDaDouble-Chain Branched Polyethylene Glycol (A Conventional Reagent)

Abbreviation: PEG₂Lys-rhIFN-β

To 1.3 ml of a 0.97 mg/ml solution of rhIFN-β obtained in ReferenceExample 4 in 20 mmol/l phosphate buffer (pH 7.8) containing ethyleneglycol and sodium chloride was added 8.3 mg (12.5 mol per mol ofprotein) of PEG₂Lys (average molecular weight: 10,000, ShearwaterPolymers, Inc.), and the mixture was subjected to reaction a whole dayand night at 4° C. The reaction mixture was applied to Sephadex G-25column (Amersham-Pharmacia Biotech) and subjected to buffer exchangewith 20 mmol/l sodium acetate buffer (pH 6) containing ethylene glycol.The mixture was passed through CM-Sepharose F.F. column (1.4 ml,Amersham-Pharmacia Biotech), followed by washing with 20 mmol/l sodiumacetate buffer (pH 6) containing ethylene glycol. Elution was carriedout with the same buffer containing 0.1 to 0.5 mol/l sodium chloride,and the desired fractions were combined and then concentrated to obtain2.7 ml of a solution containing the desired substance (0.36 mg/ml)(yield: 76.7%).

<Electrophoresis>

SDS-PAGE was carried out in the presence of 2-mercaptoethanol in amanner similar to Example 9 to confirm the bands of 1 to 3molecules-bound substances.

<Gel Filtration HPLC Analysis>

Analysis was carried out using two TSKgelG4000SW_(XL) columns in amanner similar to Example 9.

Retention time: 45.3 minutes (1 molecule-bound substance) 41.5 minutes(2 molecules-bound substance)

REFERENCE EXAMPLE 2

Preparation of Recombinant Human Granulocyte-Colony Stimulating FactorDerivative Modified with 5 KDa Double-Chain Branched Polyethylene Glycol(a Conventional Reagent)

Abbreviation: PEG₂Lys-rhG-CSF derivative

To 0.5 ml of a 4.0 mg/ml solution of the rhG-CSF derivative obtained inReference Example 5 in 50 mmol/l phosphate buffer (pH 7.5) was added10.6 mg (10 mol per mol of protein) of PEG₂Lys (average molecularweight: 10,000, Shearwater Polymers, Inc.), and the mixture wassubjected to reaction a whole day and night at 4° C. The reactionmixture was applied to Sephadex G-25 column (Amersham-Pharmacia Biotech)and subjected to buffer exchange with 20 mmol/l sodium acetate buffer(pH 4.5). The mixture was passed through SP-Sepharose F.F. column (2.0ml, Amersham-Pharmacia Biotech), followed by washing with 10 ml of 20mmol/l sodium acetate buffer (pH 4.5). Elution was carried out with thesame buffer containing 0.2 to 0.5 mol/l sodium chloride, and the desiredfractions were combined and then concentrated to obtain 0.5 ml of asolution containing the desired substance (1.05 mg/ml) (yield: 26.3%).

<Electrophoresis>

SDS-PAGE was carried out in the absence of 2-mercaptoethanol in a mannersimilar to Example 9 to confirm the bands of 1 to 3 molecules-boundsubstances.

<Gel Filtration HPLC Analysis>

Analysis was carried out using two TSKgelG4000SW_(XL) columns in amanner similar to Example 9.

Retention time: 44.3 minutes (1 molecule-bound substance) 41.7 minutes(2 molecules-bound substance)

REFERENCE EXAMPLE 3

Preparation of Recombinant Human Granulocyte-Colony Stimulating FactorModified with 5 KDa Single-Chain Polyethylene Glycol (a ConventionalReagent)

Abbreviation: PEG₂Lys-rhG-CSF

To 0.5 ml of a 4.4 mg/ml solution of rhG-CSF obtained in ReferenceExample 6 in isotonic phosphate buffer (pH 7.4) was added 11.7 mg (10mol per mol of protein) of PEG₂Lys (average molecular weight: 10,000,Shearwater Polymers, Inc.), and the mixture was subjected to reaction awhole day and night at 4° C. The reaction mixture was applied toSephadex G-25 column (Amersham-Pharmacia Biotech) and subjected tobuffer exchange with 20 mmol/l acetate buffer (pH 4.5). The mixture waspassed through SP-Sepharose F.F. column (2.0 ml, Amersham-PharmaciaBiotech), followed by washing with 10 ml of 20 mmol/l sodium acetatebuffer (pH 4.5). Elution was carried out with the same buffer containing0.2 to 0.5 mol/l sodium chloride, and the desired fractions werecombined and then concentrated to obtain 0.5 ml of a solution containingthe desired substance (1.78 mg/ml) (yield: 40.5%).

<Electrophoresis>

SDS-PAGE was carried out in the absence of 2-mercaptoethanol in a mannersimilar to Example 9 to confirm the bands of 1 to 3 molecules-boundsubstances.

<Gel Filtration HPLC Analysis>

Analysis was carried out using two TSKgelG4000SW_(XL) columns in amanner similar to Example 9.

Retention time: 44.2 minutes (1 molecule-bound substance) 41.8 minutes(2 molecules-bound substance)

REFERENCE EXAMPLE 4

Preparation of Recombinant Human Interferon-β (Unmodified rhIFN-β)

rhIFN-β having the amino acid sequence shown in SEQ ID NO: 1 wasproduced according to the method of Mizukami, et al. [BiotechnologyLetter, Vol. 8, p. 605 (1986)] and the method of Kuga, et al. [ChemistryToday, extra number 12: Gene Engineering in Medical Science, p. 135(1986), Tokyo Kagaku Dojin].

Escherichia coli K-12 carrying plasmid pMG-1 comprising DNA encodingrhIFN-β was seed-cultured in LGTrpAp medium (10 g/l bactotrypton, 5 g/lyeast extract, 5 g/l sodium chloride, 1 g/l glucose, 50 mg/lL-tryptophan and 50 μg/l ampicillin). For the production of rhIFN-β,culturing was carried out in a 2-1 jar fermenter using MCGAp medium (amedium prepared by adding 0.5% Casamino acid and 50 μg/ml ampicillin toM9 medium) at 20° C. for several days, during which the glucoseconcentration was maintained at 1% and pH at 6.5. The culture was shakenat 750 rpm and aerated at 1 l/minute. From the culture, an extract wasprepared by the freezing and thawing method [DNA, Vol. 2, p. 265(1983)]. Further, rhIFN-β was obtained from the cell residue accordingto the method disclosed in Japanese Published Unexamined PatentApplication No. 69799/86.

REFERENCE EXAMPLE 5

Preparation of Recombinant Human Granulocyte-Colony Stimulating FactorDerivative (Unmodified rhG-CSF Derivative)

An rhG-CSF derivative wherein threonine at position 1 was replaced withalanine, leucine at position 3 was replaced with threonine, glycine atposition 4 was replaced with tyrosine, proline at position 5 wasreplaced with arginine and cysteine at position 17 was replaced withserine in hG-CSF having the amino acid sequence shown in SEQ ID NO: 2was obtained by the method described in Japanese Published ExaminedPatent Application No. 96558/95.

Escherichia coli W3110strA carrying plasmid pCfBD28 comprising DNAencoding the above rhG-CSF derivative (Escherihica coli ECfBD28 FERMBP-1479) was cultured in LG medium (a medium prepared by dissolving 10 gof bactotrypton, 5 g of yeast extract, 5 g of sodium chloride and 1 g ofglucose in 1 L of water and adjusted to pH 7.0 with NaOH) at 37° C. for18 hours. The resulting culture (5 ml) was inoculated into 100 ml of MCGmedium (0.6% Na₂HPO₄, 0.3% KH₂PO₄, 0.5% sodium chloride, 0.5% Casaminoacid, 1 mmol/l MgSO₄, 14 μg/ml vitamin B, pH 7.2) containing 25 μg/mltryptophan and 50 μg/ml ampicillin. After culturing at 30° C. for 4 to 8hours, 10 μg/ml 3-indoleacrylic acid (hereinafter abbreviated as IAA), atryptophan inducer, was added, followed by further culturing for 2 to 12hours. The obtained culture was centrifuged at 8,000 rpm for 10 minutesto collect cells, and the cells were washed with a 30 mmol/l aqueoussolution of sodium chloride and 30 mmol/l tris-hydrochloride buffer (pH7.5). The washed cells were suspended in 30 ml of the above buffer anddisrupted by ultrasonication (BRANSON SONIC POWER COMPANY, SONIFIER CELLDISRUPTOR 200, OUTPUT CONTROL 2) at 0° C. for 10 minutes. Theultrasonicated cells were centrifuged at 9,000 rpm for 30 minutes toobtain cell residue.

From the cell residue, the rhG-CSF derivative was extracted, purified,solubilized and regenerated in accordance with the method of Marston, etal. [BIO/TECHNOLOGY, Vol. 2, p. 800 (1984)].

REFERENCE EXAMPLE 6

Preparation of Recombinant Human Granulocyte-Colony Stimulating Factor(Unmodified rhG-CSF)

rhG-CSF having the amino acid sequence shown in SEQ ID NO: 2 wasprepared according to the method described in Reference Example 5.

TEST EXAMPLE 1

Antiviral Activity of Chemically Modified Interferon-β

The antiviral activity of the chemically modified rhIFN-β obtained inExamples 9, 10, 12 and 13 and unmodified rhIFN-β was examined by thefollowing neutral red (NR) uptake method.

<NR Uptake Method>

The antiviral activity was measured by referring to the method ofKohase, et al. [Protein, Nucleic Acid and Enzyme (extra number), p. 335(1981)].

That is, 5% fetal bovine serum (FBS)-supplemented Eagle's MEM was addedto a sterilized transfer plate. Then, 50 μl each of solutions ofdomestic standard IFN preparations [α (The Green Cross Corporation), β(Toray Industries, Inc.) and γ (The Green Cross Corporation)] were putinto wells, followed by 2-fold serial dilution. On the other hand, 50 μleach of chemically modified IFNs and unmodified IFNs diluted with amedium to predetermined concentrations were put into wells. These IFNsolutions were transferred to a 96-well plate containing a predeterminedcell number of an established cell line (FL cell) derived from humanamnion, followed by stirring for several seconds. The resulting mixtureswere incubated a whole day and night in a CO₂ incubator at 37° C. toinduce an antiviral state.

Then, the culture liquors were removed, and a virus solution was added,followed by incubation in a CO₂ incubator at 37° C. for 2 days to effectviral infection. The antiviral state of the cells was changed by IFN,and cell degeneration occurred. Subsequently, the culture liquors wereremoved, and a neutral red (NR) solution was added. The plate wasallowed to stand in a CO₂ incubator at 37° C. for one hour, followed byremoval of the NR solution. After the wells were washed with an isotonicphosphate buffer, an extracting liquid (0.01 mol/l hydrochloric acid—30%ethanol) was added, followed by stirring for 2 to 3 minutes.

The surviving cells were stained with NR. After extraction, theabsorbance at 492 nm was measured, and a standard curve was plotted. Therelative activity of each chemically modified IFN was calculated basedon the activity of the unmodified IFN calculated from the standard curvewhich was defined as 100%.

The relative activity of each IFN-β is shown in Tables 5 and 6. TABLE 5Antiviral activity of chemically modified recombinant human IFN-βCompound abbreviation Example Relative activity (%) Unmodified rhIFN-β —100 5TRC (3UA)-rhIFN-β 9 58 5SKA (3UA)-rhIFN-β 10 93 5PET (3UA)-rhIFN-β12 50

TABLE 6 Antiviral activity of chemically modified recombinant human¹⁷Ser IFN-β Compound abbreviation Example Relative activity (%)Unmodified ¹⁷Ser rhIFN-β — 100 5PET (3UA)-¹⁷Ser rhIFN-β 13 115

It was confirmed by the results in Tables 5 and 6 that all thechemically modified IFN-β according to the present invention retainedantiviral activity.

TEST EXAMPLE 2

Antiviral Activity of Chemically Modified Interferon-α

The antiviral activity of the chemically modified rhIFN-α obtained inExample 14 and unmodified rhIFN-α was examined by the NR uptake methodillustrated in Test Example 1.

The activity of each IFN-α at a concentration of 1 Ag/ml is shown inTable 7 (indicated as a relative activity based on the activity ofunmodified IFN-α defined as 100%). TABLE 7 Antiviral activity ofchemically modified recombinant human IFN-α Compound ConcentrationRelative abbreviation Example (μg/ml) activity (%) Unmodified rhIFN-α —1 100 5PET (3UA)-rhIFN-α 14 1 100

TEST EXAMPLE 3

Growth-Promoting Activity of Chemically Modified Recombinant HumanGranulocyte-Colony Stimulating Factor Derivative on Mouse Leukemia CellNFS60

The growth-promoting activity of the compounds of Examples 15 to 20,unmodified rhG-CSF derivative and unmodified rhG-CSF on mouse leukemiacell NFS60 [Proc.

Natl. Acad. Sci. USA, Vol. 82, p. 6687 (1985)] was measured according tothe method of Asano, et al. [Japanese Pharmacology & Therapeutics, Vol.19, p. 2767 (1991)].

The activity of each compound at a concentration of 100 ng/ml is shownin Tables 8 and 9 as a relative activity based on the activity ofunmodified polypeptide defined as 100%. TABLE 8 NFS60 cellgrowth-promoting activity of chemically modified rhG-CSF derivativesConcentration Relative Compound abbreviation Example (ng/ml) activity(%) Unmodified rhG-CSF deriv. — 100 100 5SKA (3UA)-rhG-CSF deriv. 15 100100 5QNA (4UA)-rhG-CSF deriv. 16 100 100 5PET (3UU)-rhG-CSF deriv. 18100 100 5PET (3UA)-rhG-CSF deriv. 19 100 100 5SUG (3UA)-rhG-CSF deriv.20 100 100

TABLE 9 NFS60 cell growth-promoting activity of chemically modifiedrhG-CSF Concentration Relative Compound abbreviation Example (ng/ml)activity (%) Unmodified rhG-CSF — 100 100 5SKA (3UA)-rhG-CSF 17 100 100

It was confirmed by the results in Tables 8 and 9 that all thechemically modified rhG-CSF derivatives and chemically modified rhG-CSFaccording to the present invention retained growth-promoting activity onNFS60 cells.

TEST EXAMPLE 4

Enzyme Activity of Chemically Modified Superoxide Dismutase The enzymeactivity of the chemically modified SOD prepared in Example 21 wasmeasured by the xanthine-xanthine oxidase-cytochrome C system of Mccord,J. M. and Fridovichi, I. [J. Biol. Chem., Vol. 244, p. 6049 (1969)]. Oneunit (U) of SOD activity is an enzyme amount of SOD which inhibits thereducing rate of cytochrome C by 50% at pH 7.8 at 30° C., and wascalculated according to the following equation.${{Specific}\quad{activity}\quad\left( {U\text{/}{mg}} \right)} = {\left( {\frac{blank}{\Delta\quad{A/{\min.}}} - 1} \right) \times \frac{1}{0.000256}}$

The enzyme activity of chemically modified human SOD is shown in Table10.

SOD 50 U/ml=0.000256 mg (at 3900 U/mg)

ΔA/min.: measurement result TABLE 10 Enzyme activity of chemicallymodified human Cu, Zn-superoxide dismutase Compound Example Relativeactivity (%) Unmodified hSOD — 100 5PET (3UM)-hSOD 21 50*The activity was indicated as a relative activity based on the enzymeactivity of unmodified hSOD defined as 100%.

It was confirmed by Table 10 that chemically modified hSOD according tothe present invention retained enzyme activity.

TEST EXAMPLE 5

Binding Activity of Chemically Modified Anti-GD3 Chimera Antibody

The binding activity of the chemically modified anti-GD3 chimeraantibody (5PET(3UA)-KM871) prepared in Example 22 was measured accordingto the method of Kenya. S, et al. [Cancer Immunol. Immunother., Vol. 36,p. 373 (1993)].

The GD3-binding activity of unmodified antibody and chemically modifiedanti-GD3 chimera antibody (5PET(3UA)-KM871) at a concentration of 3.3g/ml is shown in Table 11.

The activity was indicated as a relative activity based on the bindingactivity of unmodified anti-GD3 chimera antibody defined as 100%. TABLE11 GD3-Binding activity of chemically modified antibody Relative bindingCompound Example activity (%) Unmodified antibody — 100 5PET (3UA)-KM87122 86.3

It was confirmed by Table 11 that the chemically modified anti-GD3chimera antibody (5PET(3UA)-KM871) according to the present inventionretained GD3-binding activity.

TEST EXAMPLE 6

Blood Half-Life Prolonging Effect of Chemically Modified Interferon-β

Each of 5TRC(3UA)-rhIFN-β obtained in Example 9, PEG₂Lys-rhIFN-βobtained in Reference Example 1 and unmodified rhIFN-β obtained inReference Example 4 was dissolved in an isotonic phosphate buffer at aconcentration of 12.5 μg/ml, and 200 μl of each of the solutions wasintravenously injected into 8 to 10-week-old BALB/C male mice (CharlesRiver Japan, Inc.). At intervals, the mice were killed and the serum wascollected. The IFN-β concentration in blood was calculated by ELISA.

The result is shown in FIG. 1.

The concentration of unmodified IFN-β fell below the detection limit inone hour after the administration, whereas the concentration ofchemically modified IFN-β was maintained for several hours, showing aremarkable improvement in durability.

Moreover, the compound disclosed in the present invention, i.e. rhIFN-βmodified with three-chain branched polyethylene glycol was superior indurability in blood to rhIFN-β modified with double-chain branchedpolyethylene glycol, and its concentration in blood changed at a higherlevel.

TEST EXAMPLE 7 Blood Half-Life Prolonging Effect of Chemically ModifiedrhG-CSF

Each of 5SKA(3UA)-rhG-CSF derivative obtained in Example 15,5SKA(3UA)-rhG-CSF obtained in Example 17, PEG₂Lys-rhG-CSF derivativeobtained in Reference Example 2, PEG₂Lys-rhG-CSF obtained in ReferenceExample 3, unmodified rhG-CSF derivative of Reference Example 5 andunmodified rhG-CSF of Reference Example 6 was intravenously injectedinto male rats at a dose of 0.1 mg/kg. At intervals, blood was collectedfrom the tail vein. The blood was appropriately diluted and theconcentration of each compound in the blood was measured by ELISA. Theresult obtained by duplicate experiments is shown in FIG. 2.

The chemically modified G-CSFs maintained much higher concentration inblood as compared with the unmodified G-CSFs. Moreover, it was confirmedthat the compounds disclosed in the present invention, i.e. rhG-CSFsmodified with three-chain branched polyethylene glycol were superior indurability in blood to the compounds modified with conventionaldouble-chain branched polyethylene glycol.

INDUSTRIAL APPLICABILITY

The novel polyalkylene glycols having a branched structure disclosed inthe present invention are useful as chemical modifying reagents forphysiologically active polypeptides. The physiologically active peptidesmodified with the polyalkylene glycols not only retain biologicalactivities similar to those of unmodified peptides, but show theirphysiological activities effectively for a long time when administeredinto the body. Therefore, the modified polypeptides are useful forimproving or treating clinical conditions associated with theirphysiological activities.

1. A branched polyalkylene glycol wherein three or more single-chainpolyalkylene glycols and a group having reactivity with an amino acidside chain, the N-terminal amino group or the C-terminal carboxyl groupin a polypeptide or a group convertible into the group having reactivityare bound.
 2. A branched polyalkylene glycol represented by formula (1):(R¹—M_(n)—X¹)_(m)L(X²—X³—R²)_(q)  (I) {wherein L represents a groupcapable of having four or more branches; M represents OCH₂CH₂,OCH₂CH₂CH₂, OCH(CH₃)CH₂, (OCH²CH²)_(r)—(OCH₂CH₂CH₂)_(s) (in which r ands, which may be the same or different, each represent an arbitrarypositive integer) or (OCH²CH²)_(ra)—[OCH(CH₃)CH₂]_(sa) (in which ra andsa have the same meanings as the above r and s, respectively); nrepresents an arbitrary positive integer; m represents an integer of 3or more; q represents an integer of 1 to 3; R¹ represents a hydrogenatom, lower alkyl or lower alkanoyl; R² represents a group havingreactivity with an amino acid side chain, the N-terminal amino group orthe C-terminal carboxyl group in a polypeptide or a group convertibleinto the group having reactivity; X¹ represents a bond, O, S, alkylene,O(CH₂)_(ta) (in which ta represents an integer of 1 to 8), (CH₂)_(tb)O(in which tb has the same meaning as the above ta), NR³ (in which R³represents a hydrogen atom or lower alkyl), R⁴—NH—C(═O)—R⁵ [in which R⁴represents a bond, alkylene or O(CH₂)_(tc) (in which tc has the samemeaning as the above ta) and R⁵ represents a bond, alkylene or OR^(5a)(in which R^(5a) represents a bond or alkylene)], R⁶—C(═O)—NH—R⁷ [inwhich R⁶ represents a bond, alkylene or R^(6a)O (in which R^(6a) has thesame meaning as the above R^(5a)) and R⁷ represents a bond, alkylene or(CH₂)_(td)O (in which td has the same meaning as the above ta)],R⁸—C(═O)—O (in which R⁸ has the same meaning as the above R^(5a)) orO—C(═O)—R⁹ (in which R⁹ has the same meaning as the above R^(5a)); X²represents a bond, O or (CH₂)_(te)O (in which te has the same meaning asthe above ta); X³ represents a bond or alkylene; and three or moreR¹—M_(n)—X¹ 's may be the same or different, and when two or threeX²—X³-R²'s are present (when q is 2 or 3), they may be the same ordifferent}.
 3. The branched polyalkylene glycol according to claim 2,wherein q is
 1. 4. The branched polyalkylene glycol according to claim2, wherein m is 3 or
 4. 5. The branched polyalkylene glycol according toany of claims 2 to 4, wherein n is 10 to 100,000, and r and s, and raand sa, which may be the same or different, each represent 1 to 100,000.6. The branched polyalkylene glycol according to claim 5, wherein R² isa hydroxyl group, carboxy, formyl, amino, vinylsulfonyl, mercapto,cyano, carbamoyl, halogenated carbonyl, halogenated lower alkyl,isocyanato, isothiocyanato, oxiranyl, lower alkanoyloxy, maleimido,succinimidooxycarbonyl, substituted or unsubstituted aryloxycarbonyl,benzotriazolyloxycarbonyl, phthalim idooxycarbonyl, imidazolylcarbonyl,substituted or unsubstituted lower alkoxycarbonyloxy, substituted orunsubstituted aryloxycarbonyloxy, tresyl, lower alkanoyloxycarbonyl,substituted or unsubstituted aroyloxycarbonyl, substituted orunsubstituted aryldisulfido, or azido.
 7. The branched polyalkyleneglycol according to claim 6, which has a molecular weight of 500 to1,000,000.
 8. The branched polyalkylene glycol according to claim 7,wherein L is a group selected from the group consisting of a groupformed by removing four or more hydrogen atoms from tricine, a groupformed by removing four or more hydrogen atoms from shikimic acid, agroup formed by removing four or more hydrogen atoms from quinic acid, agroup formed by removing four or more hydrogen atoms from erythritol, agroup formed by removing four or more hydrogen atoms frompentaerythritol, and a group formed by removing four or more hydrogenatoms from glucose.
 9. A chemically modified polypeptide wherein aphysiologically active polypeptide or its derivative is modified with atleast one branched polyalkylene glycol according to claim 8 directly orthrough a spacer.
 10. The chemically modified polypeptide according toclaim 9, wherein the physiologically active polypeptide is an enzyme, acytokine or a hormone.
 11. A pharmaceutical composition comprising thechemically modified polypeptide according to claim 10 and apharmaceutically acceptable courier.